Some Useful Packages

#If you haven't installed them here you go:
#install.packages("NACHO")
#install.packages('nanostringr')
library(BiocManager)
#BiocManager::install("GEOquery")
#BiocManager::install("limma")
#load them up:
library(NACHO)
library(nanostringr)
library(ggsci)
library(ggplot2)
library(gridExtra)
library(limma)
library(data.table)
library(png)
library(dplyr)
library(phyloseq)
library(DESeq2)
library(ComplexHeatmap)
pal1 <- pal_simpsons("springfield")(16) #makes a color palette for the figures

Data Import and Normalization

Here we read in the .RCC count files for all of the miRNA microarray assays in the lab currently. We will subset this later to just Usher and NBT cell lines. Here we use the NACHO package to normalize the data. The top houskeeping miRNAs are predicted by NACHO (housekeeping_predict = T) using the function:

find_housekeeping <- function(data, id_colname, count_column) {
  CodeClass <- NULL # no visible binding for global variable
  data <- data[grep("Endogenous|Housekeeping", CodeClass)]
  ratios <- lapply(
    X = split(data, data[[id_colname]]),
    count_column = count_column,
    FUN = function(.data, count_column) {
      .data <- .data[j = .SD, .SDcols = c("Name", count_column)]
      sample_means <- mean(.data[[count_column]])
      ratios <- log2(.data[[count_column]] / sample_means)
      names(ratios) <- .data[["Name"]]
      ratios[is.infinite(ratios)] <- NA_real_
      ratios
    }
  )
  ratios <- do.call("cbind", ratios)
  ratiosd <- apply(X = ratios, MARGIN = 1, FUN = stats::sd, na.rm = TRUE)
  ratiosd <- sort(ratiosd, decreasing = FALSE)
  names(ratiosd)[1:5]
}

and the count data are normalized by their respective geometric mean (normalisation_method = “GEO”) using the following:

geometric_mean <- function(x) {
  x[x == 0] <- 1
  exp(mean(log(x)))
}

“The normalisation methods GEOor GLM are used to compute the normalisation factors for positive and housekeeping probes. They also used to compute the”negative factor” (poor naming choice though): “geometric mean” (GEO) or the corresponding value from GLM of negative probes.

To summarise NACHO computes a data.frame with three columns:

“Positive_factor” “Negative_factor” “House_factor” (not necessarily used) The simple formula for the normalisation is: (Count_i - geo(Negative_i)) * PositiveF (with PositiveF: mean(geo(Positive)) / geo(Positive_i)) or (Count - Negative) * PositiveF * HousekeepingF In addition negative counts are replaced with 0.

For short, background is taken into account. Although, you can add additional threshold to be more “biologically relevant”.

If you are interested in details:

R/norm_geo.R R/norm_glm.R geometric_housekeeping.R factor_calculation.R normalise_counts.R”

ush_mirna_nacho <-load_rcc(data_directory = "rcc_files/",
         ssheet_csv = "samplesheet.csv",
         id_colname = "IDFILE",
         housekeeping_predict = T)
## [NACHO] Importing RCC files.
## [NACHO] Performing QC and formatting data.
## [NACHO] Searching for the best housekeeping genes.
## [NACHO] Computing normalisation factors using "GEO" method for housekeeping genes prediction.
## [NACHO] The following predicted housekeeping genes will be used for normalisation:
##   - hsa-miR-664b-5p
##   - hsa-miR-887-3p
##   - hsa-miR-29c-3p
##   - hsa-miR-373-3p
##   - hsa-miR-3150b-3p
## [NACHO] Computing normalisation factors using "GEO" method.
## [NACHO] Normalising data using "GEO" method with housekeeping genes.
## [NACHO] Returning a list.
##   $ access              : character
##   $ housekeeping_genes  : character
##   $ housekeeping_predict: logical
##   $ housekeeping_norm   : logical
##   $ normalisation_method: character
##   $ remove_outliers     : logical
##   $ n_comp              : numeric
##   $ data_directory      : character
##   $ pc_sum              : data.frame
##   $ nacho               : data.frame
##   $ outliers_thresholds : list
ush_norm <- normalise(
  nacho_object = ush_mirna_nacho,#the imported data
  #housekeeping_genes = housekeeping, #can provide a list of housekeeping genes if preferred
  housekeeping_predict = T, #predict good housekeeping candidates
  housekeeping_norm = TRUE, #normalize relative to predicted housekeeping genes
  normalisation_method = "GEO", #geometric mean calculation
  remove_outliers = F #you can specify whether to keep or get rid of outliers that did not make the QC cutoff for one reason or another...
)
## [NACHO] Nothing was done. Parameters in "normalise()", were the same as in "ush_mirna_nacho".
#visualise(ush_norm)
#render(ush_norm)

Extract the data that we want to look at, in this case the sample data and the count data:

#Select phenotype data that you want, in our case this takes the columns from our sample data (SD)
#that match what we want to look at, and makes a data.table object of them calls selected_pheno:
selected_pheno <- ush_norm[["nacho"]][
  j = lapply(unique(.SD), function(x) ifelse(x == "NA", NA, x)),
  .SDcols = c("IDFILE", "sampleID", "cartridgeID", "phenotype","genotype","source","USHvsCONT","Immortal_vs_fresh","carrier_vs_nbt","cell_source","cell_status","origin","study")
]
selected_pheno <- na.exclude(selected_pheno) #remove any data from the data table that has n/a or empty information

#get normalised cout data from our "nacho" object
expr_counts <- ush_norm[["nacho"]][
  i = grepl("Endogenous", CodeClass),
  j = as.matrix(
    dcast(.SD, Name ~ IDFILE, value.var = "Count_Norm"),
    "Name"
  ),
  .SDcols = c("IDFILE", "Name", "Count_Norm")
]

samplekept <- intersect(selected_pheno[["IDFILE"]], colnames(expr_counts))
expr_counts <- expr_counts[,samplekept]
selected_pheno <- selected_pheno[IDFILE %in% c(samplekept)]

#write.table(expr_counts, file = "all_miRNA_array.norm.epr.cts.tsv", sep="\t")

Now is the point where we will subset the dataset into the samples of interest, which are the Usher phenotypes and our NBT control phenotypes transformed with EBV.

#subset dataset to match comparisons desired....
#we need last 2 runs Usher samples plus controls -NBTi from newest run...
#could reach out to Jennifer at UNMC and see how she goes about combining runs...

ush_vs_cont_pheno <- selected_pheno[selected_pheno$USHvsCONT %in% c("yes"),]
ush_vs_cont_samplekept <- intersect(ush_vs_cont_pheno[["IDFILE"]], colnames(expr_counts))
ush_vs_cont_expr_counts <- expr_counts[,ush_vs_cont_samplekept]
rownames(ush_vs_cont_pheno) <- ush_vs_cont_pheno$sampleID
colnames(ush_vs_cont_expr_counts) <- ush_vs_cont_pheno$sampleID


#make some phyloseq objects

uvc_tax <- data.frame(ush_vs_cont_expr_counts)
uvc_tax$Kingdom <- paste0(rownames(uvc_tax))
uvc_tax$Phylum <- paste0(rownames(uvc_tax))
uvc_tax$Class <- paste0(rownames(uvc_tax))
uvc_tax$Order <-paste0(rownames(uvc_tax))
uvc_tax$Family <- paste0(rownames(uvc_tax))
uvc_tax$Genus <- paste0(rownames(uvc_tax))
uvc_tax$Species <- paste0(rownames(uvc_tax))
uvc_tax2 <- uvc_tax[,c(18:24)]
uvc_tax2 <- tax_table(as(uvc_tax2,"matrix"))


uvc.otu <- otu_table(ush_vs_cont_expr_counts,taxa_are_rows = T)

uvc.samdat<- sample_data(ush_vs_cont_pheno[,c(2,1,3:13)])
rownames(uvc.samdat) <- uvc.samdat$sampleID

uvc.ps <- phyloseq(uvc.otu,uvc.samdat,uvc_tax2)
uvc.ps
## phyloseq-class experiment-level object
## otu_table()   OTU Table:         [ 798 taxa and 17 samples ]
## sample_data() Sample Data:       [ 17 samples by 13 sample variables ]
## tax_table()   Taxonomy Table:    [ 798 taxa by 7 taxonomic ranks ]

PERMANOVA Analysis

This test will tell us if our phenotype miRNA profiles are significantly different from one another, here we use the variable sample type.

library(vegan)
set.seed(1234)
metadat <- data.frame(sample_data(uvc.ps))

permanova <- adonis2(phyloseq::distance(uvc.ps, method="bray") ~ phenotype + source, data= metadat, permutation=999)
permanova
## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Permutation: free
## Number of permutations: 999
## 
## adonis2(formula = phyloseq::distance(uvc.ps, method = "bray") ~ phenotype + source, data = metadat, permutations = 999)
##           Df SumOfSqs      R2      F Pr(>F)    
## phenotype  3  0.66613 0.82682 20.688  0.001 ***
## Residual  13  0.13953 0.17318                  
## Total     16  0.80565 1.00000                  
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
permanova$Model <- row.names(permanova)
permanova <-permanova[,c(6,1:5)]
library(microViz)
library(ggrepel)
library(gridExtra)
# first we make a function that replaces any unwanted "_" in our taxa labels with spaces
library(stringr) 
renamer <- function(x) str_replace(x, pattern = "hsa-miR-", replacement = "")

pca <- uvc.ps %>% microViz::ord_calc(method = "PCA")
pca_ord <- ord_plot(data = pca,
                           color = "phenotype",
                           shape = "phenotype",
                           plot_taxa = 1:8,
                           taxon_renamer = renamer,
                           tax_vec_length = 1.5, # this value is a scalar multiplier for the biplot score vectors
    tax_lab_style = tax_lab_style(size = 2, 
                                  alpha = 0.8, 
                                  type = "text", 
                                  max_angle = 90,
                                  check_overlap = T), # create a list of options to tweak the taxa labels' default style
    constraint_vec_style = vec_constraint(size = 0.5, alpha = 0.75), # this styles the constraint arrows
    constraint_lab_style = constraint_lab_style(size = 1, justify = "auto"),
    size = 4) + # this styles the constrain
  scale_color_manual(values = pal1) +
  coord_fixed(ratio = 1, clip = "off", xlim = c(-400,500),ylim = c(-200,450))+
  scale_shape_manual(name = "Cell Line Sample Type",
                       #breaks = c("NBT_A","USHtype1","USHtype2","USHtype3"),
                       labels = c("Control","USH1", "USH2","USH3"),
                       values = c(19,17,17,17)) +
  scale_color_manual(name = "Cell Line Sample Type",
                       #breaks = c("NBT_A","USHtype1","USHtype2","USHtype3"),
                       labels = c("Control","USH1", "USH2","USH3"),
                       values = c("#FED439FF", "#709AE1FF", "#8A9197FF", "#91331FFF")) + 
  theme(plot.title = element_text(hjust = 0.5),
        panel.border = element_rect(colour ="gray", fill = NA, size = .5),
        plot.background = element_rect(colour = "gray", fill = NA, size = 2))
pca_ord

tt1 <- ttheme_minimal(core=list(bg_params = list(fill = "white",col=NA),
                                fg_params=list(fontface=3, fontsize=6)),
                      colhead=list(fg_params=list(fontface=4L, fontsize = 6),
                                   bg_params = list(fill = "white")))
perm <- tableGrob(permanova, rows=NULL, theme = tt1)
library(gtable)
library(grid)
perm <- gtable_add_grob(perm,
        grobs = rectGrob(gp = gpar(fill = NA, lwd = 2)),
        t = 2, b = nrow(perm), l = 1, r = ncol(perm))
perm <- gtable_add_grob(perm,
        grobs = rectGrob(gp = gpar(fill = NA, lwd = 2)),
        t = 1, l = 1, r = ncol(perm))
pca_ord_perm <- pca_ord + annotation_custom(perm, 
                    xmin=-450, xmax=100, ymin=250, ymax=500)
pca_ord_perm

#ggsave(filename = "PCA_permanova_usher_miRNA.pdf",pca_ord_perm,device = "pdf", width = 8, height = 5.5)

DESeq2 diff miRNAs by phenotype:

library("DESeq2")#;packageVersion('DESeq2')

gm_mean = function(x, na.rm=TRUE){
  exp(sum(log(x[x > 0]), na.rm=na.rm) / length(x))
}
dds <- phyloseq_to_deseq2(uvc.ps, ~phenotype)
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
geoMeans <- apply(counts(dds), 1, gm_mean)
dds = estimateSizeFactors(dds,geoMeans=geoMeans)

dds = DESeq2::DESeq(dds, test = "Wald", fitType = "local")
## using pre-existing size factors
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## final dispersion estimates
## fitting model and testing
resultsNames(dds)
## [1] "Intercept"                   "phenotype_USHtype1_vs_NBT_A"
## [3] "phenotype_USHtype2_vs_NBT_A" "phenotype_USHtype3_vs_NBT_A"
#########################################################################################################################
ush1.nbt.res = results(dds, contrast =c("phenotype","USHtype1","NBT_A") )
ush1.nbt.res = ush1.nbt.res[order(ush1.nbt.res$padj, na.last=NA), ]
alpha = 0.05

ush1.nbt.sigtab = ush1.nbt.res[(ush1.nbt.res$padj < alpha), ]
ush1.nbt.sigtab = cbind(as(ush1.nbt.sigtab, "data.frame"), as(tax_table(uvc.ps)[rownames(ush1.nbt.sigtab), ], "matrix"))

#order df by log fold change and manually inspect where 2fold change threshold is, in this case row 76:
o = order(abs(ush1.nbt.sigtab$log2FoldChange), decreasing = TRUE)
ush1.nbt.sigtab2 = ush1.nbt.sigtab[o, ]
ush1.nbt.sigtab2 = ush1.nbt.sigtab2[1:63,]
ush1.nbt.sigtab2
##                                     baseMean log2FoldChange     lfcSE
## hsa-miR-222-3p                     62.352476       8.976366 1.0862126
## hsa-miR-151a-5p                    12.943991      -7.652711 1.4140075
## hsa-miR-424-5p                     26.056195       7.140241 1.5007809
## hsa-miR-503-5p                     22.185375       6.870487 1.0943090
## hsa-miR-129-2-3p                    6.247654      -6.585901 1.8476038
## hsa-miR-1252-5p                    14.162051       6.541487 1.1172016
## hsa-miR-1246                     1161.454061      -6.441675 0.4618959
## hsa-miR-182-5p                     13.386544      -6.269056 1.7802903
## hsa-miR-1205                       11.550695       6.250660 1.1192896
## hsa-miR-450a-5p                    22.279584       5.927644 1.5799898
## hsa-miR-654-5p                      5.891644       5.915866 1.2486557
## hsa-miR-519d-3p                     3.246283       5.575579 1.6558303
## hsa-miR-4431                        2.400438       5.438074 1.9621137
## hsa-miR-345-5p                      5.074769      -5.414299 1.3203787
## hsa-miR-3934-5p                     5.185416       5.396254 1.2974910
## hsa-miR-221-5p                      4.368518       5.309392 1.6473012
## hsa-miR-152-3p                      3.702954      -5.218438 1.4915337
## hsa-miR-1260b                       2.641261       4.969649 1.7505240
## hsa-miR-34a-5p                    454.895501       4.897727 0.4509239
## hsa-miR-150-5p                    452.972299       4.890114 0.6106738
## hsa-let-7d-5p                     588.882373       4.811113 0.3670308
## hsa-miR-483-3p                      7.946179       4.715437 1.1509874
## hsa-miR-146b-5p                   168.632116       4.652693 0.4681016
## hsa-miR-371b-5p                     3.811814       4.622638 1.3699610
## hsa-miR-601                         6.414515      -4.573882 1.2045490
## hsa-miR-5010-3p                     3.921794       4.453129 1.4213647
## hsa-miR-591                         6.943709       4.275723 1.2521012
## hsa-miR-155-5p                  11097.869650       4.149979 0.2869964
## hsa-miR-363-3p                    319.076852       4.128234 0.3272720
## hsa-miR-3916                        4.334913      -3.864626 1.2674039
## hsa-let-7i-5p                    1348.149768       3.814222 0.3545566
## hsa-miR-1827                        6.679916       3.800521 1.0123302
## hsa-miR-4455                       14.743625      -3.777094 1.1651531
## hsa-miR-3195                        3.248441      -3.771340 1.4107073
## hsa-miR-195-5p                      2.898308      -3.751481 1.5077286
## hsa-miR-1226-3p                     4.558990       3.710526 1.3886886
## hsa-miR-27a-3p                      3.104900       3.700705 1.5096094
## hsa-miR-194-5p                     42.517210      -3.379095 0.4028265
## hsa-miR-28-3p                      59.451504      -3.358252 0.4224829
## hsa-miR-151a-3p                    10.199230      -3.351741 0.7765824
## hsa-miR-10a-5p                     44.111464       3.055764 0.4277549
## hsa-miR-146a-5p                  4963.645408       2.989872 0.4023580
## hsa-miR-337-3p                     54.247834       2.981863 0.4663584
## hsa-miR-200c-3p                    16.731190       2.942878 0.7288186
## hsa-miR-96-5p                      34.142701      -2.938594 0.3424560
## hsa-miR-5196-3p+hsa-miR-6732-3p    14.758482       2.777010 0.6499589
## hsa-miR-551b-3p                    33.524252       2.734946 0.4275855
## hsa-miR-299-3p                     27.522827      -2.675616 0.7050000
## hsa-let-7f-5p                     763.539513       2.652028 0.3456879
## hsa-miR-577                         4.625677      -2.605055 0.9970668
## hsa-miR-181b-2-3p                   7.627180       2.530375 0.8285307
## hsa-let-7g-5p                    2774.856795       2.442113 0.3122241
## hsa-miR-1287-5p                     7.496055       2.357994 0.9396846
## hsa-miR-324-5p                     48.937752      -2.290057 0.7169758
## hsa-miR-484                        46.995584      -2.287798 0.3329308
## hsa-miR-183-5p                     36.030068      -2.277099 0.3826565
## hsa-miR-582-5p                     32.434430      -2.265008 0.6650003
## hsa-let-7b-5p                     108.579356       2.203120 0.2818926
## hsa-miR-16-5p                    9716.681454      -2.184471 0.3741371
## hsa-miR-331-3p                    216.410641      -2.176946 0.3457068
## hsa-miR-148a-3p                   543.177533       2.173471 0.3608197
## hsa-miR-98-5p                     271.663474       2.170957 0.3540343
## hsa-miR-26b-5p                    457.008546      -2.124356 0.3741439
##                                       stat       pvalue         padj
## hsa-miR-222-3p                    8.263913 1.409735e-16 6.653947e-15
## hsa-miR-151a-5p                  -5.412072 6.229962e-08 7.738269e-07
## hsa-miR-424-5p                    4.757684 1.958272e-06 2.009357e-05
## hsa-miR-503-5p                    6.278379 3.421215e-10 6.728389e-09
## hsa-miR-129-2-3p                 -3.564563 3.644624e-04 2.047931e-03
## hsa-miR-1252-5p                   5.855243 4.763138e-09 7.494004e-08
## hsa-miR-1246                    -13.946163 3.319940e-44 7.835059e-42
## hsa-miR-182-5p                   -3.521367 4.293277e-04 2.356310e-03
## hsa-miR-1205                      5.584488 2.343891e-08 3.253873e-07
## hsa-miR-450a-5p                   3.751698 1.756413e-04 1.105369e-03
## hsa-miR-654-5p                    4.737788 2.160636e-06 2.124625e-05
## hsa-miR-519d-3p                   3.367241 7.592437e-04 3.853366e-03
## hsa-miR-4431                      2.771539 5.579206e-03 2.330430e-02
## hsa-miR-345-5p                   -4.100565 4.121427e-05 2.903453e-04
## hsa-miR-3934-5p                   4.158991 3.196558e-05 2.357462e-04
## hsa-miR-221-5p                    3.223085 1.268179e-03 6.107965e-03
## hsa-miR-152-3p                   -3.498706 4.675222e-04 2.536442e-03
## hsa-miR-1260b                     2.838950 4.526230e-03 1.959982e-02
## hsa-miR-34a-5p                   10.861538 1.757632e-27 1.659205e-25
## hsa-miR-150-5p                    8.007735 1.168402e-15 5.013506e-14
## hsa-let-7d-5p                    13.108201 2.955238e-39 4.649575e-37
## hsa-miR-483-3p                    4.096863 4.187874e-05 2.906877e-04
## hsa-miR-146b-5p                   9.939493 2.802494e-23 1.889682e-21
## hsa-miR-371b-5p                   3.374285 7.400778e-04 3.796921e-03
## hsa-miR-601                      -3.797174 1.463552e-04 9.671047e-04
## hsa-miR-5010-3p                   3.132995 1.730323e-03 8.167125e-03
## hsa-miR-591                       3.414838 6.381987e-04 3.346998e-03
## hsa-miR-155-5p                   14.460040 2.166726e-47 1.022695e-44
## hsa-miR-363-3p                   12.614077 1.766155e-36 2.084062e-34
## hsa-miR-3916                     -3.049246 2.294166e-03 1.051307e-02
## hsa-let-7i-5p                    10.757725 5.449920e-27 4.287270e-25
## hsa-miR-1827                      3.754231 1.738747e-04 1.105369e-03
## hsa-miR-4455                     -3.241715 1.188127e-03 5.877339e-03
## hsa-miR-3195                     -2.673368 7.509375e-03 3.016351e-02
## hsa-miR-195-5p                   -2.488167 1.284034e-02 4.556872e-02
## hsa-miR-1226-3p                   2.671964 7.540878e-03 3.016351e-02
## hsa-miR-27a-3p                    2.451432 1.422889e-02 4.974842e-02
## hsa-miR-194-5p                   -8.388462 4.925435e-17 2.583117e-15
## hsa-miR-28-3p                    -7.948847 1.882553e-15 7.404710e-14
## hsa-miR-151a-3p                  -4.316015 1.588711e-05 1.249786e-04
## hsa-miR-10a-5p                    7.143728 9.083316e-13 2.381847e-11
## hsa-miR-146a-5p                   7.430874 1.078828e-13 3.182542e-12
## hsa-miR-337-3p                    6.393931 1.616741e-10 3.468645e-09
## hsa-miR-200c-3p                   4.037874 5.393782e-05 3.689660e-04
## hsa-miR-96-5p                    -8.580940 9.410125e-18 5.551974e-16
## hsa-miR-5196-3p+hsa-miR-6732-3p   4.272593 1.932130e-05 1.495025e-04
## hsa-miR-551b-3p                   6.396254 1.592346e-10 3.468645e-09
## hsa-miR-299-3p                   -3.795200 1.475244e-04 9.671047e-04
## hsa-let-7f-5p                     7.671740 1.696780e-14 5.339201e-13
## hsa-miR-577                      -2.612718 8.982532e-03 3.505201e-02
## hsa-miR-181b-2-3p                 3.054051 2.257739e-03 1.044758e-02
## hsa-let-7g-5p                     7.821669 5.212766e-15 1.846314e-13
## hsa-miR-1287-5p                   2.509346 1.209549e-02 4.358071e-02
## hsa-miR-324-5p                   -3.194051 1.402914e-03 6.688638e-03
## hsa-miR-484                      -6.871692 6.344478e-12 1.576102e-10
## hsa-miR-183-5p                   -5.950765 2.668923e-09 4.343903e-08
## hsa-miR-582-5p                   -3.406025 6.591607e-04 3.418944e-03
## hsa-let-7b-5p                     7.815458 5.476356e-15 1.846314e-13
## hsa-miR-16-5p                    -5.838691 5.261246e-09 8.010672e-08
## hsa-miR-331-3p                   -6.297089 3.032879e-10 6.223995e-09
## hsa-miR-148a-3p                   6.023706 1.704680e-09 2.980033e-08
## hsa-miR-98-5p                     6.132053 8.675229e-10 1.574888e-08
## hsa-miR-26b-5p                   -5.677913 1.363481e-08 1.950191e-07
##                                                         Kingdom
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-151a-5p                                 hsa-miR-151a-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-129-2-3p                               hsa-miR-129-2-3p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-519d-3p                                 hsa-miR-519d-3p
## hsa-miR-4431                                       hsa-miR-4431
## hsa-miR-345-5p                                   hsa-miR-345-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-152-3p                                   hsa-miR-152-3p
## hsa-miR-1260b                                     hsa-miR-1260b
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-601                                         hsa-miR-601
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-3916                                       hsa-miR-3916
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-miR-4455                                       hsa-miR-4455
## hsa-miR-3195                                       hsa-miR-3195
## hsa-miR-195-5p                                   hsa-miR-195-5p
## hsa-miR-1226-3p                                 hsa-miR-1226-3p
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-151a-3p                                 hsa-miR-151a-3p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-181b-2-3p                             hsa-miR-181b-2-3p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-1287-5p                                 hsa-miR-1287-5p
## hsa-miR-324-5p                                   hsa-miR-324-5p
## hsa-miR-484                                         hsa-miR-484
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-16-5p                                     hsa-miR-16-5p
## hsa-miR-331-3p                                   hsa-miR-331-3p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-26b-5p                                   hsa-miR-26b-5p
##                                                          Phylum
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-151a-5p                                 hsa-miR-151a-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-129-2-3p                               hsa-miR-129-2-3p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-519d-3p                                 hsa-miR-519d-3p
## hsa-miR-4431                                       hsa-miR-4431
## hsa-miR-345-5p                                   hsa-miR-345-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-152-3p                                   hsa-miR-152-3p
## hsa-miR-1260b                                     hsa-miR-1260b
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-601                                         hsa-miR-601
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-3916                                       hsa-miR-3916
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-miR-4455                                       hsa-miR-4455
## hsa-miR-3195                                       hsa-miR-3195
## hsa-miR-195-5p                                   hsa-miR-195-5p
## hsa-miR-1226-3p                                 hsa-miR-1226-3p
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-151a-3p                                 hsa-miR-151a-3p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-181b-2-3p                             hsa-miR-181b-2-3p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-1287-5p                                 hsa-miR-1287-5p
## hsa-miR-324-5p                                   hsa-miR-324-5p
## hsa-miR-484                                         hsa-miR-484
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-16-5p                                     hsa-miR-16-5p
## hsa-miR-331-3p                                   hsa-miR-331-3p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-26b-5p                                   hsa-miR-26b-5p
##                                                           Class
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-151a-5p                                 hsa-miR-151a-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-129-2-3p                               hsa-miR-129-2-3p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-519d-3p                                 hsa-miR-519d-3p
## hsa-miR-4431                                       hsa-miR-4431
## hsa-miR-345-5p                                   hsa-miR-345-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-152-3p                                   hsa-miR-152-3p
## hsa-miR-1260b                                     hsa-miR-1260b
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-601                                         hsa-miR-601
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-3916                                       hsa-miR-3916
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-miR-4455                                       hsa-miR-4455
## hsa-miR-3195                                       hsa-miR-3195
## hsa-miR-195-5p                                   hsa-miR-195-5p
## hsa-miR-1226-3p                                 hsa-miR-1226-3p
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-151a-3p                                 hsa-miR-151a-3p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-181b-2-3p                             hsa-miR-181b-2-3p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-1287-5p                                 hsa-miR-1287-5p
## hsa-miR-324-5p                                   hsa-miR-324-5p
## hsa-miR-484                                         hsa-miR-484
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-16-5p                                     hsa-miR-16-5p
## hsa-miR-331-3p                                   hsa-miR-331-3p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-26b-5p                                   hsa-miR-26b-5p
##                                                           Order
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-151a-5p                                 hsa-miR-151a-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-129-2-3p                               hsa-miR-129-2-3p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-519d-3p                                 hsa-miR-519d-3p
## hsa-miR-4431                                       hsa-miR-4431
## hsa-miR-345-5p                                   hsa-miR-345-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-152-3p                                   hsa-miR-152-3p
## hsa-miR-1260b                                     hsa-miR-1260b
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-601                                         hsa-miR-601
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-3916                                       hsa-miR-3916
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-miR-4455                                       hsa-miR-4455
## hsa-miR-3195                                       hsa-miR-3195
## hsa-miR-195-5p                                   hsa-miR-195-5p
## hsa-miR-1226-3p                                 hsa-miR-1226-3p
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-151a-3p                                 hsa-miR-151a-3p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-181b-2-3p                             hsa-miR-181b-2-3p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-1287-5p                                 hsa-miR-1287-5p
## hsa-miR-324-5p                                   hsa-miR-324-5p
## hsa-miR-484                                         hsa-miR-484
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-16-5p                                     hsa-miR-16-5p
## hsa-miR-331-3p                                   hsa-miR-331-3p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-26b-5p                                   hsa-miR-26b-5p
##                                                          Family
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-151a-5p                                 hsa-miR-151a-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-129-2-3p                               hsa-miR-129-2-3p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-519d-3p                                 hsa-miR-519d-3p
## hsa-miR-4431                                       hsa-miR-4431
## hsa-miR-345-5p                                   hsa-miR-345-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-152-3p                                   hsa-miR-152-3p
## hsa-miR-1260b                                     hsa-miR-1260b
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-601                                         hsa-miR-601
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-3916                                       hsa-miR-3916
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-miR-4455                                       hsa-miR-4455
## hsa-miR-3195                                       hsa-miR-3195
## hsa-miR-195-5p                                   hsa-miR-195-5p
## hsa-miR-1226-3p                                 hsa-miR-1226-3p
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-151a-3p                                 hsa-miR-151a-3p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-181b-2-3p                             hsa-miR-181b-2-3p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-1287-5p                                 hsa-miR-1287-5p
## hsa-miR-324-5p                                   hsa-miR-324-5p
## hsa-miR-484                                         hsa-miR-484
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-16-5p                                     hsa-miR-16-5p
## hsa-miR-331-3p                                   hsa-miR-331-3p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-26b-5p                                   hsa-miR-26b-5p
##                                                           Genus
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-151a-5p                                 hsa-miR-151a-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-129-2-3p                               hsa-miR-129-2-3p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-519d-3p                                 hsa-miR-519d-3p
## hsa-miR-4431                                       hsa-miR-4431
## hsa-miR-345-5p                                   hsa-miR-345-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-152-3p                                   hsa-miR-152-3p
## hsa-miR-1260b                                     hsa-miR-1260b
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-601                                         hsa-miR-601
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-3916                                       hsa-miR-3916
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-miR-4455                                       hsa-miR-4455
## hsa-miR-3195                                       hsa-miR-3195
## hsa-miR-195-5p                                   hsa-miR-195-5p
## hsa-miR-1226-3p                                 hsa-miR-1226-3p
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-151a-3p                                 hsa-miR-151a-3p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-181b-2-3p                             hsa-miR-181b-2-3p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-1287-5p                                 hsa-miR-1287-5p
## hsa-miR-324-5p                                   hsa-miR-324-5p
## hsa-miR-484                                         hsa-miR-484
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-16-5p                                     hsa-miR-16-5p
## hsa-miR-331-3p                                   hsa-miR-331-3p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-26b-5p                                   hsa-miR-26b-5p
##                                                         Species
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-151a-5p                                 hsa-miR-151a-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-129-2-3p                               hsa-miR-129-2-3p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-519d-3p                                 hsa-miR-519d-3p
## hsa-miR-4431                                       hsa-miR-4431
## hsa-miR-345-5p                                   hsa-miR-345-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-152-3p                                   hsa-miR-152-3p
## hsa-miR-1260b                                     hsa-miR-1260b
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-601                                         hsa-miR-601
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-3916                                       hsa-miR-3916
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-miR-4455                                       hsa-miR-4455
## hsa-miR-3195                                       hsa-miR-3195
## hsa-miR-195-5p                                   hsa-miR-195-5p
## hsa-miR-1226-3p                                 hsa-miR-1226-3p
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-151a-3p                                 hsa-miR-151a-3p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-181b-2-3p                             hsa-miR-181b-2-3p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-1287-5p                                 hsa-miR-1287-5p
## hsa-miR-324-5p                                   hsa-miR-324-5p
## hsa-miR-484                                         hsa-miR-484
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-16-5p                                     hsa-miR-16-5p
## hsa-miR-331-3p                                   hsa-miR-331-3p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-26b-5p                                   hsa-miR-26b-5p
#########################################################################################################################
ush2.nbt.res = results(dds, contrast =c("phenotype","USHtype2","NBT_A") )
ush2.nbt.res = ush2.nbt.res[order(ush2.nbt.res$padj, na.last=NA), ]
ush2.nbt.sigtab = ush2.nbt.res[(ush2.nbt.res$padj < alpha), ]
ush2.nbt.sigtab = cbind(as(ush2.nbt.sigtab, "data.frame"), as(tax_table(uvc.ps)[rownames(ush2.nbt.sigtab), ], "matrix"))

#order df by log fold change and manually inspect where 2fold change threshold is, in this case row 76:
o = order(abs(ush2.nbt.sigtab$log2FoldChange), decreasing = TRUE)
ush2.nbt.sigtab2 = ush2.nbt.sigtab[o, ]
ush2.nbt.sigtab2 = ush2.nbt.sigtab2[1:62,]
ush2.nbt.sigtab2
##                                     baseMean log2FoldChange     lfcSE      stat
## hsa-miR-150-5p                    452.972299       9.778649 0.6146488 15.909327
## hsa-miR-222-3p                     62.352476       8.870729 1.0944227  8.105396
## hsa-miR-424-5p                     26.056195       8.157197 1.5433530  5.285373
## hsa-miR-182-5p                     13.386544      -8.037607 1.9945504 -4.029784
## hsa-miR-503-5p                     22.185375       7.681362 1.1005214  6.979748
## hsa-miR-450a-5p                    22.279584       7.297461 1.6271085  4.484926
## hsa-miR-1252-5p                    14.162051       7.074685 1.1272707  6.275942
## hsa-miR-1205                       11.550695       6.445804 1.1330601  5.688845
## hsa-miR-146b-5p                   168.632116       6.088318 0.4802753 12.676724
## hsa-let-7d-5p                     588.882373       5.638169 0.3829947 14.721274
## hsa-miR-574-3p                      3.881123       5.375367 1.5182670  3.540462
## hsa-miR-34a-5p                    454.895501       5.311438 0.4715676 11.263365
## hsa-miR-513b-5p                     4.472696      -5.288974 1.5553773 -3.400444
## hsa-miR-483-3p                      7.946179       5.196772 1.1651060  4.460343
## hsa-miR-3934-5p                     5.185416       5.101567 1.3401854  3.806613
## hsa-miR-654-5p                      5.891644       5.045064 1.2956111  3.893965
## hsa-miR-137                         3.212125      -4.918130 1.5168913 -3.242243
## hsa-miR-223-3p                    159.998728       4.803963 0.4137021 11.612131
## hsa-miR-221-5p                      4.368518       4.752997 1.7215001  2.760962
## hsa-miR-28-3p                      59.451504      -4.730881 0.5026720 -9.411468
## hsa-miR-155-5p                  11097.869650       4.641151 0.3023737 15.349056
## hsa-miR-5010-3p                     3.921794       4.593808 1.4696719  3.125737
## hsa-let-7i-5p                    1348.149768       4.591980 0.3728104 12.317198
## hsa-miR-591                         6.943709       4.531152 1.2811069  3.536904
## hsa-miR-370-3p                      7.208119      -4.505703 1.2364461 -3.644075
## hsa-miR-503-3p                      3.150306       4.501648 1.6023650  2.809377
## hsa-miR-577                         4.625677      -4.182267 1.2823862 -3.261316
## hsa-miR-146a-5p                  4963.645408       3.645911 0.4239996  8.598854
## hsa-miR-98-5p                     271.663474       3.591315 0.3694847  9.719795
## hsa-miR-767-5p                     11.339515      -3.547808 0.8331848 -4.258128
## hsa-let-7g-5p                    2774.856795       3.535712 0.3288041 10.753248
## hsa-miR-200c-3p                    16.731190       3.532417 0.7472186  4.727421
## hsa-miR-1246                     1161.454061      -3.461864 0.4797713 -7.215653
## hsa-miR-363-3p                    319.076852       3.349837 0.3439406  9.739580
## hsa-miR-96-5p                      34.142701      -3.270057 0.3932114 -8.316283
## hsa-miR-132-3p                    128.534206       3.121741 0.4182450  7.463904
## hsa-miR-194-5p                     42.517210      -3.012283 0.4281448 -7.035664
## hsa-let-7b-5p                     108.579356       2.885862 0.2926133  9.862373
## hsa-let-7f-5p                     763.539513       2.770965 0.3640103  7.612326
## hsa-miR-183-5p                     36.030068      -2.679509 0.4253317 -6.299810
## hsa-miR-1183                       14.842391      -2.677247 0.6562571 -4.079570
## hsa-miR-148a-3p                   543.177533       2.670289 0.3797355  7.031972
## hsa-miR-28-5p                      61.342319      -2.573117 0.4597829 -5.596373
## hsa-miR-542-5p                     10.002203       2.563074 0.7582140  3.380409
## hsa-let-7e-5p                       9.166687       2.553093 0.7141781  3.574868
## hsa-miR-23c                        12.349246      -2.420985 0.7402905 -3.270317
## hsa-miR-337-3p                     54.247834       2.391787 0.4919785  4.861568
## hsa-miR-5196-3p+hsa-miR-6732-3p    14.758482       2.372361 0.6845475  3.465590
## hsa-miR-551b-3p                    33.524252       2.320197 0.4506935  5.148060
## hsa-miR-10a-5p                     44.111464       2.318068 0.4528395  5.118962
## hsa-miR-299-3p                     27.522827      -2.209553 0.7432988 -2.972631
## hsa-miR-221-3p                     18.166067       2.201322 0.8422616  2.613584
## hsa-miR-374b-5p                   119.433283       2.147022 0.4597624  4.669851
## hsa-miR-23a-3p                    414.360207       2.131325 0.2983583  7.143508
## hsa-let-7c-5p                      41.178953       2.119217 0.3306348  6.409541
## hsa-miR-1304-5p                    14.303492       2.107587 0.5285978  3.987127
## hsa-miR-514a-3p                    14.397073      -2.079384 0.6107492 -3.404645
## hsa-let-7a-5p                    3774.101892       2.076836 0.3213073  6.463705
## hsa-miR-342-3p                    345.669854       2.057311 0.3992218  5.153304
## hsa-miR-181a-5p                   961.247540      -2.034583 0.4444699 -4.577551
## hsa-miR-181c-5p                    21.150719      -2.027716 0.5632067 -3.600306
## hsa-miR-1193                       21.196875       2.010495 0.4499887  4.467878
##                                       pvalue         padj
## hsa-miR-150-5p                  5.459739e-57 2.647974e-54
## hsa-miR-222-3p                  5.257388e-16 1.699889e-14
## hsa-miR-424-5p                  1.254486e-07 1.789487e-06
## hsa-miR-182-5p                  5.582820e-05 5.014200e-04
## hsa-miR-503-5p                  2.957100e-12 6.235624e-11
## hsa-miR-450a-5p                 7.293916e-06 7.861220e-05
## hsa-miR-1252-5p                 3.475246e-10 6.242572e-09
## hsa-miR-1205                    1.279012e-08 2.215431e-07
## hsa-miR-146b-5p                 7.958962e-37 9.650242e-35
## hsa-let-7d-5p                   4.707086e-49 7.609790e-47
## hsa-miR-574-3p                  3.994268e-04 2.887511e-03
## hsa-miR-34a-5p                  1.990115e-29 1.378865e-27
## hsa-miR-513b-5p                 6.727649e-04 4.531819e-03
## hsa-miR-483-3p                  8.182875e-06 8.444030e-05
## hsa-miR-3934-5p                 1.408829e-04 1.138804e-03
## hsa-miR-654-5p                  9.861905e-05 8.246593e-04
## hsa-miR-137                     1.185929e-03 7.374049e-03
## hsa-miR-223-3p                  3.575911e-31 2.890528e-29
## hsa-miR-221-5p                  5.763134e-03 3.071560e-02
## hsa-miR-28-3p                   4.892560e-21 1.977410e-19
## hsa-miR-155-5p                  3.593791e-53 8.714944e-51
## hsa-miR-5010-3p                 1.773601e-03 1.061971e-02
## hsa-let-7i-5p                   7.319374e-35 7.099793e-33
## hsa-miR-591                     4.048469e-04 2.887511e-03
## hsa-miR-370-3p                  2.683551e-04 2.133643e-03
## hsa-miR-503-3p                  4.963743e-03 2.735699e-02
## hsa-miR-577                     1.108963e-03 6.985028e-03
## hsa-miR-146a-5p                 8.051598e-18 3.003865e-16
## hsa-miR-98-5p                   2.482818e-22 1.094697e-20
## hsa-miR-767-5p                  2.061458e-05 1.999614e-04
## hsa-let-7g-5p                   5.721061e-27 3.468393e-25
## hsa-miR-200c-3p                 2.273890e-06 2.757091e-05
## hsa-miR-1246                    5.367563e-13 1.370141e-11
## hsa-miR-363-3p                  2.043964e-22 9.913224e-21
## hsa-miR-96-5p                   9.076483e-17 3.144353e-15
## hsa-miR-132-3p                  8.399554e-14 2.263213e-12
## hsa-miR-194-5p                  1.983141e-12 4.489229e-11
## hsa-let-7b-5p                   6.060009e-23 3.265672e-21
## hsa-let-7f-5p                   2.692060e-14 7.680289e-13
## hsa-miR-183-5p                  2.980105e-10 5.559043e-09
## hsa-miR-1183                    4.511901e-05 4.128815e-04
## hsa-miR-148a-3p                 2.036351e-12 4.489229e-11
## hsa-miR-28-5p                   2.188825e-08 3.660622e-07
## hsa-miR-542-5p                  7.237793e-04 4.743688e-03
## hsa-let-7e-5p                   3.504045e-04 2.614556e-03
## hsa-miR-23c                     1.074270e-03 6.855541e-03
## hsa-miR-337-3p                  1.164597e-06 1.448281e-05
## hsa-miR-5196-3p+hsa-miR-6732-3p 5.290697e-04 3.665697e-03
## hsa-miR-551b-3p                 2.631947e-07 3.545817e-06
## hsa-miR-10a-5p                  3.072225e-07 4.027106e-06
## hsa-miR-299-3p                  2.952593e-03 1.746350e-02
## hsa-miR-221-3p                  8.959795e-03 4.302476e-02
## hsa-miR-374b-5p                 3.014182e-06 3.480663e-05
## hsa-miR-23a-3p                  9.097836e-13 2.206225e-11
## hsa-let-7c-5p                   1.459588e-10 2.831600e-09
## hsa-miR-1304-5p                 6.687834e-05 5.897453e-04
## hsa-miR-514a-3p                 6.625002e-04 4.525530e-03
## hsa-let-7a-5p                   1.021703e-10 2.064691e-09
## hsa-miR-342-3p                  2.559368e-07 3.545817e-06
## hsa-miR-181a-5p                 4.704513e-06 5.306253e-05
## hsa-miR-181c-5p                 3.178431e-04 2.427007e-03
## hsa-miR-1193                    7.899920e-06 8.329264e-05
##                                                         Kingdom
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-513b-5p                                 hsa-miR-513b-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-137                                         hsa-miR-137
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-370-3p                                   hsa-miR-370-3p
## hsa-miR-503-3p                                   hsa-miR-503-3p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-767-5p                                   hsa-miR-767-5p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-132-3p                                   hsa-miR-132-3p
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-1183                                       hsa-miR-1183
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-23c                                         hsa-miR-23c
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-374b-5p                                 hsa-miR-374b-5p
## hsa-miR-23a-3p                                   hsa-miR-23a-3p
## hsa-let-7c-5p                                     hsa-let-7c-5p
## hsa-miR-1304-5p                                 hsa-miR-1304-5p
## hsa-miR-514a-3p                                 hsa-miR-514a-3p
## hsa-let-7a-5p                                     hsa-let-7a-5p
## hsa-miR-342-3p                                   hsa-miR-342-3p
## hsa-miR-181a-5p                                 hsa-miR-181a-5p
## hsa-miR-181c-5p                                 hsa-miR-181c-5p
## hsa-miR-1193                                       hsa-miR-1193
##                                                          Phylum
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-513b-5p                                 hsa-miR-513b-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-137                                         hsa-miR-137
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-370-3p                                   hsa-miR-370-3p
## hsa-miR-503-3p                                   hsa-miR-503-3p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-767-5p                                   hsa-miR-767-5p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-132-3p                                   hsa-miR-132-3p
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-1183                                       hsa-miR-1183
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-23c                                         hsa-miR-23c
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-374b-5p                                 hsa-miR-374b-5p
## hsa-miR-23a-3p                                   hsa-miR-23a-3p
## hsa-let-7c-5p                                     hsa-let-7c-5p
## hsa-miR-1304-5p                                 hsa-miR-1304-5p
## hsa-miR-514a-3p                                 hsa-miR-514a-3p
## hsa-let-7a-5p                                     hsa-let-7a-5p
## hsa-miR-342-3p                                   hsa-miR-342-3p
## hsa-miR-181a-5p                                 hsa-miR-181a-5p
## hsa-miR-181c-5p                                 hsa-miR-181c-5p
## hsa-miR-1193                                       hsa-miR-1193
##                                                           Class
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-513b-5p                                 hsa-miR-513b-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-137                                         hsa-miR-137
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-370-3p                                   hsa-miR-370-3p
## hsa-miR-503-3p                                   hsa-miR-503-3p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-767-5p                                   hsa-miR-767-5p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-132-3p                                   hsa-miR-132-3p
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-1183                                       hsa-miR-1183
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-23c                                         hsa-miR-23c
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-374b-5p                                 hsa-miR-374b-5p
## hsa-miR-23a-3p                                   hsa-miR-23a-3p
## hsa-let-7c-5p                                     hsa-let-7c-5p
## hsa-miR-1304-5p                                 hsa-miR-1304-5p
## hsa-miR-514a-3p                                 hsa-miR-514a-3p
## hsa-let-7a-5p                                     hsa-let-7a-5p
## hsa-miR-342-3p                                   hsa-miR-342-3p
## hsa-miR-181a-5p                                 hsa-miR-181a-5p
## hsa-miR-181c-5p                                 hsa-miR-181c-5p
## hsa-miR-1193                                       hsa-miR-1193
##                                                           Order
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-513b-5p                                 hsa-miR-513b-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-137                                         hsa-miR-137
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-370-3p                                   hsa-miR-370-3p
## hsa-miR-503-3p                                   hsa-miR-503-3p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-767-5p                                   hsa-miR-767-5p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-132-3p                                   hsa-miR-132-3p
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-1183                                       hsa-miR-1183
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-23c                                         hsa-miR-23c
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-374b-5p                                 hsa-miR-374b-5p
## hsa-miR-23a-3p                                   hsa-miR-23a-3p
## hsa-let-7c-5p                                     hsa-let-7c-5p
## hsa-miR-1304-5p                                 hsa-miR-1304-5p
## hsa-miR-514a-3p                                 hsa-miR-514a-3p
## hsa-let-7a-5p                                     hsa-let-7a-5p
## hsa-miR-342-3p                                   hsa-miR-342-3p
## hsa-miR-181a-5p                                 hsa-miR-181a-5p
## hsa-miR-181c-5p                                 hsa-miR-181c-5p
## hsa-miR-1193                                       hsa-miR-1193
##                                                          Family
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-513b-5p                                 hsa-miR-513b-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-137                                         hsa-miR-137
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-370-3p                                   hsa-miR-370-3p
## hsa-miR-503-3p                                   hsa-miR-503-3p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-767-5p                                   hsa-miR-767-5p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-132-3p                                   hsa-miR-132-3p
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-1183                                       hsa-miR-1183
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-23c                                         hsa-miR-23c
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-374b-5p                                 hsa-miR-374b-5p
## hsa-miR-23a-3p                                   hsa-miR-23a-3p
## hsa-let-7c-5p                                     hsa-let-7c-5p
## hsa-miR-1304-5p                                 hsa-miR-1304-5p
## hsa-miR-514a-3p                                 hsa-miR-514a-3p
## hsa-let-7a-5p                                     hsa-let-7a-5p
## hsa-miR-342-3p                                   hsa-miR-342-3p
## hsa-miR-181a-5p                                 hsa-miR-181a-5p
## hsa-miR-181c-5p                                 hsa-miR-181c-5p
## hsa-miR-1193                                       hsa-miR-1193
##                                                           Genus
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-513b-5p                                 hsa-miR-513b-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-137                                         hsa-miR-137
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-370-3p                                   hsa-miR-370-3p
## hsa-miR-503-3p                                   hsa-miR-503-3p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-767-5p                                   hsa-miR-767-5p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-132-3p                                   hsa-miR-132-3p
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-1183                                       hsa-miR-1183
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-23c                                         hsa-miR-23c
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-374b-5p                                 hsa-miR-374b-5p
## hsa-miR-23a-3p                                   hsa-miR-23a-3p
## hsa-let-7c-5p                                     hsa-let-7c-5p
## hsa-miR-1304-5p                                 hsa-miR-1304-5p
## hsa-miR-514a-3p                                 hsa-miR-514a-3p
## hsa-let-7a-5p                                     hsa-let-7a-5p
## hsa-miR-342-3p                                   hsa-miR-342-3p
## hsa-miR-181a-5p                                 hsa-miR-181a-5p
## hsa-miR-181c-5p                                 hsa-miR-181c-5p
## hsa-miR-1193                                       hsa-miR-1193
##                                                         Species
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-513b-5p                                 hsa-miR-513b-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-137                                         hsa-miR-137
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-370-3p                                   hsa-miR-370-3p
## hsa-miR-503-3p                                   hsa-miR-503-3p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-767-5p                                   hsa-miR-767-5p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-132-3p                                   hsa-miR-132-3p
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-1183                                       hsa-miR-1183
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-23c                                         hsa-miR-23c
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-374b-5p                                 hsa-miR-374b-5p
## hsa-miR-23a-3p                                   hsa-miR-23a-3p
## hsa-let-7c-5p                                     hsa-let-7c-5p
## hsa-miR-1304-5p                                 hsa-miR-1304-5p
## hsa-miR-514a-3p                                 hsa-miR-514a-3p
## hsa-let-7a-5p                                     hsa-let-7a-5p
## hsa-miR-342-3p                                   hsa-miR-342-3p
## hsa-miR-181a-5p                                 hsa-miR-181a-5p
## hsa-miR-181c-5p                                 hsa-miR-181c-5p
## hsa-miR-1193                                       hsa-miR-1193
########################################################################################################################

ush3.nbt.res = results(dds, contrast =c("phenotype","USHtype3","NBT_A") )
ush3.nbt.res = ush3.nbt.res[order(ush3.nbt.res$padj, na.last=NA), ]
ush3.nbt.sigtab = ush3.nbt.res[(ush3.nbt.res$padj < alpha), ]
ush3.nbt.sigtab = cbind(as(ush3.nbt.sigtab, "data.frame"), as(tax_table(uvc.ps)[rownames(ush3.nbt.sigtab), ], "matrix"))

#order df by log fold change and manually inspect where 2fold change threshold is, in this case row 76:
o = order(abs(ush3.nbt.sigtab$log2FoldChange), decreasing = TRUE)
ush3.nbt.sigtab2 = ush3.nbt.sigtab[o, ]
ush3.nbt.sigtab2 = ush3.nbt.sigtab2[1:56,]
ush3.nbt.sigtab2
##                                     baseMean log2FoldChange     lfcSE
## hsa-miR-222-3p                     62.352476       8.567235 1.0947828
## hsa-miR-503-5p                     22.185375       7.436675 1.1010052
## hsa-miR-424-5p                     26.056195       7.282262 1.5458130
## hsa-miR-150-5p                    452.972299       6.589296 0.6167416
## hsa-miR-1205                       11.550695       6.513342 1.1306670
## hsa-miR-450a-5p                    22.279584       6.427184 1.6296560
## hsa-miR-1252-5p                    14.162051       6.397175 1.1320479
## hsa-miR-182-5p                     13.386544      -6.173477 1.9068428
## hsa-miR-125a-3p                     2.918606       5.702255 1.9135982
## hsa-miR-574-3p                      3.881123       5.627804 1.5118064
## hsa-miR-1246                     1161.454061      -5.543254 0.4841454
## hsa-miR-27a-3p                      3.104900       5.520852 1.5172155
## hsa-miR-34a-5p                    454.895501       5.496695 0.4713569
## hsa-miR-483-3p                      7.946179       5.466798 1.1590900
## hsa-miR-146b-5p                   168.632116       5.403200 0.4810898
## hsa-miR-5010-3p                     3.921794       5.387762 1.4469663
## hsa-let-7d-5p                     588.882373       5.316598 0.3830935
## hsa-miR-3934-5p                     5.185416       5.178786 1.3348461
## hsa-miR-654-5p                      5.891644       4.997846 1.2928424
## hsa-miR-221-5p                      4.368518       4.724120 1.7185689
## hsa-miR-155-5p                  11097.869650       4.689762 0.3023658
## hsa-miR-28-3p                      59.451504      -4.685299 0.4928530
## hsa-let-7i-5p                    1348.149768       4.396787 0.3728220
## hsa-miR-577                         4.625677      -4.362689 1.2866443
## hsa-miR-371b-5p                     3.811814       4.300270 1.4156369
## hsa-miR-200c-3p                    16.731190       3.999709 0.7407163
## hsa-miR-98-5p                     271.663474       3.674355 0.3692115
## hsa-miR-591                         6.943709       3.637626 1.2949258
## hsa-miR-146a-5p                  4963.645408       3.567453 0.4239950
## hsa-miR-363-3p                    319.076852       3.381946 0.3436155
## hsa-miR-601                         6.414515      -3.372557 1.1293441
## hsa-miR-194-5p                     42.517210      -3.323784 0.4329041
## hsa-miR-10a-5p                     44.111464       3.261432 0.4429706
## hsa-let-7g-5p                    2774.856795       3.241588 0.3288241
## hsa-miR-223-3p                    159.998728       3.234347 0.4163864
## hsa-miR-1827                        6.679916       3.175454 1.0533972
## hsa-let-7f-5p                     763.539513       3.114388 0.3637491
## hsa-miR-494-3p                      6.840844      -3.053108 1.0902364
## hsa-miR-337-3p                     54.247834       2.875717 0.4877725
## hsa-miR-96-5p                      34.142701      -2.740837 0.3653291
## hsa-miR-125b-5p                    21.478559      -2.710956 0.8135606
## hsa-miR-542-5p                     10.002203       2.610852 0.7551828
## hsa-miR-582-5p                     32.434430      -2.502524 0.7101236
## hsa-miR-148a-3p                   543.177533       2.491279 0.3797552
## hsa-miR-4284                      100.234448      -2.415085 0.5077947
## hsa-miR-221-3p                     18.166067       2.359329 0.8392596
## hsa-miR-183-5p                     36.030068      -2.346714 0.4115839
## hsa-miR-1183                       14.842391      -2.308941 0.6181953
## hsa-miR-181a-2-3p                  12.501246       2.285039 0.7036593
## hsa-miR-5196-3p+hsa-miR-6732-3p    14.758482       2.195847 0.6849704
## hsa-miR-374b-5p                   119.433283       2.189624 0.4593008
## hsa-let-7e-5p                       9.166687       2.139527 0.7199022
## hsa-miR-28-5p                      61.342319      -2.093236 0.4506749
## hsa-let-7b-5p                     108.579356       2.064284 0.2958303
## hsa-miR-23c                        12.349246      -1.974785 0.6984797
## hsa-miR-23a-3p                    414.360207       1.958638 0.2983817
##                                       stat       pvalue         padj
## hsa-miR-222-3p                    7.825511 5.055984e-15 1.704589e-13
## hsa-miR-503-5p                    6.754441 1.433866e-11 3.222784e-10
## hsa-miR-424-5p                    4.710959 2.465534e-06 3.232589e-05
## hsa-miR-150-5p                   10.684047 1.208836e-26 8.151011e-25
## hsa-miR-1205                      5.760619 8.380606e-09 1.521402e-07
## hsa-miR-450a-5p                   3.943890 8.017044e-05 7.568090e-04
## hsa-miR-1252-5p                   5.650976 1.595395e-08 2.689380e-07
## hsa-miR-182-5p                   -3.237539 1.205656e-03 7.295765e-03
## hsa-miR-125a-3p                   2.979860 2.883802e-03 1.495774e-02
## hsa-miR-574-3p                    3.722569 1.972058e-04 1.551353e-03
## hsa-miR-1246                    -11.449563 2.363332e-30 2.230985e-28
## hsa-miR-27a-3p                    3.638805 2.739056e-04 2.020054e-03
## hsa-miR-34a-5p                   11.661430 2.006445e-31 2.367606e-29
## hsa-miR-483-3p                    4.716457 2.399872e-06 3.232589e-05
## hsa-miR-146b-5p                  11.231166 2.866686e-29 2.255127e-27
## hsa-miR-5010-3p                   3.723488 1.964893e-04 1.551353e-03
## hsa-let-7d-5p                    13.878070 8.602975e-44 2.030302e-41
## hsa-miR-3934-5p                   3.879688 1.045907e-04 9.406923e-04
## hsa-miR-654-5p                    3.865781 1.107342e-04 9.678986e-04
## hsa-miR-221-5p                    2.748869 5.980134e-03 2.822623e-02
## hsa-miR-155-5p                   15.510228 2.958286e-54 1.396311e-51
## hsa-miR-28-3p                    -9.506484 1.972160e-21 8.462360e-20
## hsa-let-7i-5p                    11.793261 4.228466e-32 6.652786e-30
## hsa-miR-577                      -3.390750 6.970165e-04 4.569330e-03
## hsa-miR-371b-5p                   3.037693 2.383970e-03 1.293372e-02
## hsa-miR-200c-3p                   5.399786 6.672047e-08 1.085933e-06
## hsa-miR-98-5p                     9.951896 2.474224e-23 1.459792e-21
## hsa-miR-591                       2.809139 4.967426e-03 2.417139e-02
## hsa-miR-146a-5p                   8.413903 3.965876e-17 1.439918e-15
## hsa-miR-363-3p                    9.842240 7.404394e-23 3.494874e-21
## hsa-miR-601                      -2.986297 2.823779e-03 1.480915e-02
## hsa-miR-194-5p                   -7.677875 1.617490e-14 4.771597e-13
## hsa-miR-10a-5p                    7.362638 1.803112e-13 4.728159e-12
## hsa-let-7g-5p                     9.858123 6.322031e-23 3.315554e-21
## hsa-miR-223-3p                    7.767657 7.995091e-15 2.515789e-13
## hsa-miR-1827                      3.014488 2.574131e-03 1.365157e-02
## hsa-let-7f-5p                     8.561913 1.110102e-17 4.366401e-16
## hsa-miR-494-3p                   -2.800409 5.103791e-03 2.433323e-02
## hsa-miR-337-3p                    5.895612 3.732951e-09 7.047812e-08
## hsa-miR-96-5p                    -7.502377 6.267074e-14 1.740035e-12
## hsa-miR-125b-5p                  -3.332211 8.615881e-04 5.495535e-03
## hsa-miR-542-5p                    3.457245 5.457287e-04 3.787999e-03
## hsa-miR-582-5p                   -3.524068 4.249749e-04 3.039215e-03
## hsa-miR-148a-3p                   6.560222 5.372773e-11 1.102586e-09
## hsa-miR-4284                     -4.756026 1.974410e-06 2.740945e-05
## hsa-miR-221-3p                    2.811202 4.935673e-03 2.417139e-02
## hsa-miR-183-5p                   -5.701666 1.186421e-08 2.074039e-07
## hsa-miR-1183                     -3.734971 1.877372e-04 1.527792e-03
## hsa-miR-181a-2-3p                 3.247365 1.164787e-03 7.139995e-03
## hsa-miR-5196-3p+hsa-miR-6732-3p   3.205755 1.347086e-03 7.947806e-03
## hsa-miR-374b-5p                   4.767298 1.867131e-06 2.670564e-05
## hsa-let-7e-5p                     2.971969 2.958965e-03 1.518078e-02
## hsa-miR-28-5p                    -4.644669 3.406229e-06 4.345243e-05
## hsa-let-7b-5p                     6.977935 2.995511e-12 7.069406e-11
## hsa-miR-23c                      -2.827262 4.694785e-03 2.332567e-02
## hsa-miR-23a-3p                    6.564203 5.231203e-11 1.102586e-09
##                                                         Kingdom
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-125a-3p                                 hsa-miR-125a-3p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-601                                         hsa-miR-601
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-494-3p                                   hsa-miR-494-3p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-125b-5p                                 hsa-miR-125b-5p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-4284                                       hsa-miR-4284
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-1183                                       hsa-miR-1183
## hsa-miR-181a-2-3p                             hsa-miR-181a-2-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-374b-5p                                 hsa-miR-374b-5p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-23c                                         hsa-miR-23c
## hsa-miR-23a-3p                                   hsa-miR-23a-3p
##                                                          Phylum
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-125a-3p                                 hsa-miR-125a-3p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-601                                         hsa-miR-601
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-494-3p                                   hsa-miR-494-3p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-125b-5p                                 hsa-miR-125b-5p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-4284                                       hsa-miR-4284
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-1183                                       hsa-miR-1183
## hsa-miR-181a-2-3p                             hsa-miR-181a-2-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-374b-5p                                 hsa-miR-374b-5p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-23c                                         hsa-miR-23c
## hsa-miR-23a-3p                                   hsa-miR-23a-3p
##                                                           Class
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-125a-3p                                 hsa-miR-125a-3p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-601                                         hsa-miR-601
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-494-3p                                   hsa-miR-494-3p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-125b-5p                                 hsa-miR-125b-5p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-4284                                       hsa-miR-4284
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-1183                                       hsa-miR-1183
## hsa-miR-181a-2-3p                             hsa-miR-181a-2-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-374b-5p                                 hsa-miR-374b-5p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-23c                                         hsa-miR-23c
## hsa-miR-23a-3p                                   hsa-miR-23a-3p
##                                                           Order
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-125a-3p                                 hsa-miR-125a-3p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-601                                         hsa-miR-601
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-494-3p                                   hsa-miR-494-3p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-125b-5p                                 hsa-miR-125b-5p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-4284                                       hsa-miR-4284
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-1183                                       hsa-miR-1183
## hsa-miR-181a-2-3p                             hsa-miR-181a-2-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-374b-5p                                 hsa-miR-374b-5p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-23c                                         hsa-miR-23c
## hsa-miR-23a-3p                                   hsa-miR-23a-3p
##                                                          Family
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-125a-3p                                 hsa-miR-125a-3p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-601                                         hsa-miR-601
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-494-3p                                   hsa-miR-494-3p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-125b-5p                                 hsa-miR-125b-5p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-4284                                       hsa-miR-4284
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-1183                                       hsa-miR-1183
## hsa-miR-181a-2-3p                             hsa-miR-181a-2-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-374b-5p                                 hsa-miR-374b-5p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-23c                                         hsa-miR-23c
## hsa-miR-23a-3p                                   hsa-miR-23a-3p
##                                                           Genus
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-125a-3p                                 hsa-miR-125a-3p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-601                                         hsa-miR-601
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-494-3p                                   hsa-miR-494-3p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-125b-5p                                 hsa-miR-125b-5p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-4284                                       hsa-miR-4284
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-1183                                       hsa-miR-1183
## hsa-miR-181a-2-3p                             hsa-miR-181a-2-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-374b-5p                                 hsa-miR-374b-5p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-23c                                         hsa-miR-23c
## hsa-miR-23a-3p                                   hsa-miR-23a-3p
##                                                         Species
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-125a-3p                                 hsa-miR-125a-3p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-601                                         hsa-miR-601
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-494-3p                                   hsa-miR-494-3p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-125b-5p                                 hsa-miR-125b-5p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-miR-4284                                       hsa-miR-4284
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-1183                                       hsa-miR-1183
## hsa-miR-181a-2-3p                             hsa-miR-181a-2-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-374b-5p                                 hsa-miR-374b-5p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-23c                                         hsa-miR-23c
## hsa-miR-23a-3p                                   hsa-miR-23a-3p
########################################################################################################################


ush1.ush2.common_miRNAs <- merge(ush1.nbt.sigtab2, ush2.nbt.sigtab2, by = "row.names", all = T)

ush1.ush3.common_miRNAs <- merge(ush1.nbt.sigtab2,ush3.nbt.sigtab2, by = "row.names", all = T)

ush2.ush3.common_miRNAs <- merge(ush2.nbt.sigtab2,ush3.nbt.sigtab2, by = "row.names", all = T)

row.names(ush1.ush2.common_miRNAs) <- ush1.ush2.common_miRNAs$Row.names
ush1.ush2.ush3.common_miRNAs<- merge(ush1.ush2.common_miRNAs, ush3.nbt.sigtab2, by = "row.names", all = TRUE)
## Warning in merge.data.frame(ush1.ush2.common_miRNAs, ush3.nbt.sigtab2, by =
## "row.names", : column name 'Row.names' is duplicated in the result

DESeq2 diff miRNAs by sample type source:

gm_mean = function(x, na.rm=TRUE){
  exp(sum(log(x[x > 0]), na.rm=na.rm) / length(x))
}
dds <- phyloseq_to_deseq2(uvc.ps, ~source)
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
geoMeans <- apply(counts(dds), 1, gm_mean)
dds = estimateSizeFactors(dds,geoMeans=geoMeans)

dds = DESeq2::DESeq(dds, test = "Wald", fitType = "local")
## using pre-existing size factors
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## final dispersion estimates
## fitting model and testing
## -- replacing outliers and refitting for 1 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
resultsNames(dds)
## [1] "Intercept"                 "source_patient_vs_control"
#########################################################################################################################
pat.ctrl.res = results(dds, contrast =c("source","patient","control") )
pat.ctrl.res = pat.ctrl.res[order(pat.ctrl.res$padj, na.last=NA), ]
alpha = 0.05

pat.ctrl.sigtab = pat.ctrl.res[(pat.ctrl.res$padj < alpha), ]
pat.ctrl.sigtab = cbind(as(pat.ctrl.sigtab, "data.frame"), as(tax_table(uvc.ps)[rownames(pat.ctrl.sigtab), ], "matrix"))

#order df by log fold change and manually inspect where 2fold change threshold is, in this case row 76:
o = order(abs(pat.ctrl.sigtab$log2FoldChange), decreasing = TRUE)
pat.ctrl.sigtab2 = pat.ctrl.sigtab[o, ]
pat.ctrl.sigtab2 = pat.ctrl.sigtab2[1:56,]
pat.ctrl.sigtab2
##                                     baseMean log2FoldChange     lfcSE      stat
## hsa-miR-222-3p                     62.352476       8.828492 1.0741073  8.219376
## hsa-miR-150-5p                    452.972299       8.366903 1.2131767  6.896689
## hsa-miR-424-5p                     26.056195       7.568949 1.3567689  5.578657
## hsa-miR-503-5p                     22.185375       7.330651 1.0816025  6.777584
## hsa-miR-1252-5p                    14.162051       6.685254 1.0933238  6.114615
## hsa-miR-182-5p                     13.386544      -6.662012 1.2325499 -5.405065
## hsa-miR-450a-5p                    22.279584       6.617966 1.4239369  4.647654
## hsa-miR-1205                       11.550695       6.394347 1.0879091  5.877649
## hsa-miR-146b-5p                   168.632116       5.453612 0.5079041 10.737483
## hsa-miR-654-5p                      5.891644       5.442823 1.1988795  4.539925
## hsa-let-7d-5p                     588.882373       5.260088 0.3651535 14.405140
## hsa-miR-3934-5p                     5.185416       5.246460 1.2150078  4.318046
## hsa-miR-34a-5p                    454.895501       5.230921 0.4166680 12.554171
## hsa-miR-483-3p                      7.946179       5.122809 1.1120210  4.606756
## hsa-miR-221-5p                      4.368518       4.986916 1.4833170  3.362003
## hsa-miR-5010-3p                     3.921794       4.841298 1.3101743  3.695156
## hsa-miR-574-3p                      3.881123       4.818546 1.5736489  3.062021
## hsa-miR-129-2-3p                    6.247654      -4.724839 1.3871383 -3.406177
## hsa-miR-1246                     1161.454061      -4.682589 0.7596397 -6.164224
## hsa-miR-519d-3p                     3.246283       4.577166 1.5994772  2.861664
## hsa-miR-27a-3p                      3.104900       4.502615 1.4172003  3.177119
## hsa-miR-155-5p                  11097.869650       4.488373 0.2846257 15.769391
## hsa-let-7i-5p                    1348.149768       4.270705 0.3429287 12.453624
## hsa-miR-591                         6.943709       4.200138 1.1826609  3.551431
## hsa-miR-371b-5p                     3.811814       4.072891 1.3481598  3.021074
## hsa-miR-28-3p                      59.451504      -4.024072 0.4359784 -9.229979
## hsa-miR-503-3p                      3.150306       3.784286 1.4263230  2.653176
## hsa-miR-363-3p                    319.076852       3.710825 0.3406580 10.893110
## hsa-miR-223-3p                    159.998728       3.640410 0.7427580  4.901206
## hsa-miR-1226-3p                     4.558990       3.579258 1.2747018  2.807918
## hsa-miR-200c-3p                    16.731190       3.521344 0.6964937  5.055816
## hsa-miR-146a-5p                  4963.645408       3.399748 0.3736969  9.097608
## hsa-miR-577                         4.625677      -3.358210 0.9083915 -3.696875
## hsa-miR-194-5p                     42.517210      -3.244839 0.3458610 -9.381915
## hsa-miR-98-5p                     271.663474       3.223540 0.4235403  7.610942
## hsa-miR-1827                        6.679916       3.213518 1.0058213  3.194919
## hsa-let-7g-5p                    2774.856795       3.098503 0.3433172  9.025191
## hsa-miR-513b-5p                     4.472696      -3.020751 1.0663190 -2.832878
## hsa-miR-96-5p                      34.142701      -2.960385 0.2974687 -9.951921
## hsa-miR-10a-5p                     44.111464       2.937573 0.4292533  6.843449
## hsa-let-7f-5p                     763.539513       2.844295 0.3150381  9.028417
## hsa-miR-337-3p                     54.247834       2.793187 0.4342856  6.431683
## hsa-miR-5196-3p+hsa-miR-6732-3p    14.758482       2.500156 0.5947919  4.203412
## hsa-miR-148a-3p                   543.177533       2.438972 0.3292586  7.407466
## hsa-let-7b-5p                     108.579356       2.417213 0.3111384  7.768931
## hsa-miR-183-5p                     36.030068      -2.407983 0.3356446 -7.174204
## hsa-miR-551b-3p                    33.524252       2.366812 0.4273038  5.538943
## hsa-miR-132-3p                    128.534206       2.267199 0.4635145  4.891322
## hsa-let-7e-5p                       9.166687       2.187612 0.6319375  3.461754
## hsa-miR-28-5p                      61.342319      -2.181957 0.3813464 -5.721720
## hsa-miR-221-3p                     18.166067       2.145917 0.5719838  3.751710
## hsa-miR-542-5p                     10.002203       2.100097 0.7526891  2.790126
## hsa-miR-299-3p                     27.522827      -1.999593 0.5034377 -3.971878
## hsa-miR-484                        46.995584      -1.964903 0.3030808 -6.483097
## hsa-miR-582-5p                     32.434430      -1.936904 0.4886111 -3.964102
## hsa-miR-514a-3p                    14.397073      -1.857722 0.4755330 -3.906611
##                                       pvalue         padj
## hsa-miR-222-3p                  2.045647e-16 8.654660e-15
## hsa-miR-150-5p                  5.322838e-12 1.626423e-10
## hsa-miR-424-5p                  2.423821e-08 4.761078e-07
## hsa-miR-503-5p                  1.222025e-11 3.360568e-10
## hsa-miR-1252-5p                 9.679018e-10 2.129384e-08
## hsa-miR-182-5p                  6.478500e-08 1.149411e-06
## hsa-miR-450a-5p                 3.357318e-06 4.196648e-05
## hsa-miR-1205                    4.161351e-09 8.802857e-08
## hsa-miR-146b-5p                 6.786941e-27 6.221363e-25
## hsa-miR-654-5p                  5.627421e-06 6.728438e-05
## hsa-let-7d-5p                   4.803403e-47 1.320936e-44
## hsa-miR-3934-5p                 1.574166e-05 1.766921e-04
## hsa-miR-34a-5p                  3.771101e-36 6.913685e-34
## hsa-miR-483-3p                  4.089996e-06 4.998884e-05
## hsa-miR-221-5p                  7.737925e-04 5.319823e-03
## hsa-miR-5010-3p                 2.197519e-04 1.803933e-03
## hsa-miR-574-3p                  2.198480e-03 1.374050e-02
## hsa-miR-129-2-3p                6.587942e-04 4.705673e-03
## hsa-miR-1246                    7.082954e-10 1.623177e-08
## hsa-miR-519d-3p                 4.214238e-03 2.439822e-02
## hsa-miR-27a-3p                  1.487458e-03 9.624731e-03
## hsa-miR-155-5p                  5.053461e-56 2.779403e-53
## hsa-let-7i-5p                   1.336363e-35 1.837499e-33
## hsa-miR-591                     3.831429e-04 2.886693e-03
## hsa-miR-371b-5p                 2.518797e-03 1.522350e-02
## hsa-miR-28-3p                   2.706841e-20 1.654181e-18
## hsa-miR-503-3p                  7.973823e-03 4.257867e-02
## hsa-miR-363-3p                  1.243209e-27 1.367530e-25
## hsa-miR-223-3p                  9.525029e-07 1.455213e-05
## hsa-miR-1226-3p                 4.986292e-03 2.827279e-02
## hsa-miR-200c-3p                 4.285549e-07 6.734433e-06
## hsa-miR-146a-5p                 9.234350e-20 5.078893e-18
## hsa-miR-577                     2.182696e-04 1.803933e-03
## hsa-miR-194-5p                  6.478512e-21 4.453977e-19
## hsa-miR-98-5p                   2.721058e-14 9.977213e-13
## hsa-miR-1827                    1.398699e-03 9.268488e-03
## hsa-let-7g-5p                   1.793819e-19 8.221671e-18
## hsa-miR-513b-5p                 4.613102e-03 2.642923e-02
## hsa-miR-96-5p                   2.473618e-23 1.943557e-21
## hsa-miR-10a-5p                  7.730881e-12 2.237887e-10
## hsa-let-7f-5p                   1.741725e-19 8.221671e-18
## hsa-miR-337-3p                  1.261984e-10 3.017789e-09
## hsa-miR-5196-3p+hsa-miR-6732-3p 2.629215e-05 2.728430e-04
## hsa-miR-148a-3p                 1.287355e-13 4.425281e-12
## hsa-let-7b-5p                   7.915111e-15 3.109508e-13
## hsa-miR-183-5p                  7.272906e-13 2.352999e-11
## hsa-miR-551b-3p                 3.043021e-08 5.771246e-07
## hsa-miR-132-3p                  1.001607e-06 1.488876e-05
## hsa-let-7e-5p                   5.366681e-04 3.988749e-03
## hsa-miR-28-5p                   1.054511e-08 2.148079e-07
## hsa-miR-221-3p                  1.756327e-04 1.509343e-03
## hsa-miR-542-5p                  5.268753e-03 2.956953e-02
## hsa-miR-299-3p                  7.130835e-05 6.761999e-04
## hsa-miR-484                     8.985848e-11 2.246462e-09
## hsa-miR-582-5p                  7.367260e-05 6.867785e-04
## hsa-miR-514a-3p                 9.359970e-05 8.579972e-04
##                                                         Kingdom
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-129-2-3p                               hsa-miR-129-2-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-519d-3p                                 hsa-miR-519d-3p
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-503-3p                                   hsa-miR-503-3p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-1226-3p                                 hsa-miR-1226-3p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-513b-5p                                 hsa-miR-513b-5p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-132-3p                                   hsa-miR-132-3p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-miR-484                                         hsa-miR-484
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-miR-514a-3p                                 hsa-miR-514a-3p
##                                                          Phylum
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-129-2-3p                               hsa-miR-129-2-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-519d-3p                                 hsa-miR-519d-3p
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-503-3p                                   hsa-miR-503-3p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-1226-3p                                 hsa-miR-1226-3p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-513b-5p                                 hsa-miR-513b-5p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-132-3p                                   hsa-miR-132-3p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-miR-484                                         hsa-miR-484
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-miR-514a-3p                                 hsa-miR-514a-3p
##                                                           Class
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-129-2-3p                               hsa-miR-129-2-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-519d-3p                                 hsa-miR-519d-3p
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-503-3p                                   hsa-miR-503-3p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-1226-3p                                 hsa-miR-1226-3p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-513b-5p                                 hsa-miR-513b-5p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-132-3p                                   hsa-miR-132-3p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-miR-484                                         hsa-miR-484
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-miR-514a-3p                                 hsa-miR-514a-3p
##                                                           Order
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-129-2-3p                               hsa-miR-129-2-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-519d-3p                                 hsa-miR-519d-3p
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-503-3p                                   hsa-miR-503-3p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-1226-3p                                 hsa-miR-1226-3p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-513b-5p                                 hsa-miR-513b-5p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-132-3p                                   hsa-miR-132-3p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-miR-484                                         hsa-miR-484
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-miR-514a-3p                                 hsa-miR-514a-3p
##                                                          Family
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-129-2-3p                               hsa-miR-129-2-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-519d-3p                                 hsa-miR-519d-3p
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-503-3p                                   hsa-miR-503-3p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-1226-3p                                 hsa-miR-1226-3p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-513b-5p                                 hsa-miR-513b-5p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-132-3p                                   hsa-miR-132-3p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-miR-484                                         hsa-miR-484
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-miR-514a-3p                                 hsa-miR-514a-3p
##                                                           Genus
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-129-2-3p                               hsa-miR-129-2-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-519d-3p                                 hsa-miR-519d-3p
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-503-3p                                   hsa-miR-503-3p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-1226-3p                                 hsa-miR-1226-3p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-513b-5p                                 hsa-miR-513b-5p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-132-3p                                   hsa-miR-132-3p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-miR-484                                         hsa-miR-484
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-miR-514a-3p                                 hsa-miR-514a-3p
##                                                         Species
## hsa-miR-222-3p                                   hsa-miR-222-3p
## hsa-miR-150-5p                                   hsa-miR-150-5p
## hsa-miR-424-5p                                   hsa-miR-424-5p
## hsa-miR-503-5p                                   hsa-miR-503-5p
## hsa-miR-1252-5p                                 hsa-miR-1252-5p
## hsa-miR-182-5p                                   hsa-miR-182-5p
## hsa-miR-450a-5p                                 hsa-miR-450a-5p
## hsa-miR-1205                                       hsa-miR-1205
## hsa-miR-146b-5p                                 hsa-miR-146b-5p
## hsa-miR-654-5p                                   hsa-miR-654-5p
## hsa-let-7d-5p                                     hsa-let-7d-5p
## hsa-miR-3934-5p                                 hsa-miR-3934-5p
## hsa-miR-34a-5p                                   hsa-miR-34a-5p
## hsa-miR-483-3p                                   hsa-miR-483-3p
## hsa-miR-221-5p                                   hsa-miR-221-5p
## hsa-miR-5010-3p                                 hsa-miR-5010-3p
## hsa-miR-574-3p                                   hsa-miR-574-3p
## hsa-miR-129-2-3p                               hsa-miR-129-2-3p
## hsa-miR-1246                                       hsa-miR-1246
## hsa-miR-519d-3p                                 hsa-miR-519d-3p
## hsa-miR-27a-3p                                   hsa-miR-27a-3p
## hsa-miR-155-5p                                   hsa-miR-155-5p
## hsa-let-7i-5p                                     hsa-let-7i-5p
## hsa-miR-591                                         hsa-miR-591
## hsa-miR-371b-5p                                 hsa-miR-371b-5p
## hsa-miR-28-3p                                     hsa-miR-28-3p
## hsa-miR-503-3p                                   hsa-miR-503-3p
## hsa-miR-363-3p                                   hsa-miR-363-3p
## hsa-miR-223-3p                                   hsa-miR-223-3p
## hsa-miR-1226-3p                                 hsa-miR-1226-3p
## hsa-miR-200c-3p                                 hsa-miR-200c-3p
## hsa-miR-146a-5p                                 hsa-miR-146a-5p
## hsa-miR-577                                         hsa-miR-577
## hsa-miR-194-5p                                   hsa-miR-194-5p
## hsa-miR-98-5p                                     hsa-miR-98-5p
## hsa-miR-1827                                       hsa-miR-1827
## hsa-let-7g-5p                                     hsa-let-7g-5p
## hsa-miR-513b-5p                                 hsa-miR-513b-5p
## hsa-miR-96-5p                                     hsa-miR-96-5p
## hsa-miR-10a-5p                                   hsa-miR-10a-5p
## hsa-let-7f-5p                                     hsa-let-7f-5p
## hsa-miR-337-3p                                   hsa-miR-337-3p
## hsa-miR-5196-3p+hsa-miR-6732-3p hsa-miR-5196-3p+hsa-miR-6732-3p
## hsa-miR-148a-3p                                 hsa-miR-148a-3p
## hsa-let-7b-5p                                     hsa-let-7b-5p
## hsa-miR-183-5p                                   hsa-miR-183-5p
## hsa-miR-551b-3p                                 hsa-miR-551b-3p
## hsa-miR-132-3p                                   hsa-miR-132-3p
## hsa-let-7e-5p                                     hsa-let-7e-5p
## hsa-miR-28-5p                                     hsa-miR-28-5p
## hsa-miR-221-3p                                   hsa-miR-221-3p
## hsa-miR-542-5p                                   hsa-miR-542-5p
## hsa-miR-299-3p                                   hsa-miR-299-3p
## hsa-miR-484                                         hsa-miR-484
## hsa-miR-582-5p                                   hsa-miR-582-5p
## hsa-miR-514a-3p                                 hsa-miR-514a-3p

Lets spend some time figuring out which DE miRNAs are unique to each phenotype. We did this in a couple ways, one is to create the Venn Diagram which shows how many DE miRNAs are unique to each group and subgroup. Then we extract the unique values all the venn clssifications and make a table of the information.

########################################################################################################################
ush1 <- row.names(ush1.nbt.sigtab2)
ush2 <- row.names(ush2.nbt.sigtab2)
ush3 <- row.names(ush3.nbt.sigtab2)
########################################################################################################################
pat.ctrl<- row.names(pat.ctrl.sigtab2)

########################################################################################################################
library(ggvenn)
ushlist <- list('Usher 1' = ush1, 'Usher 2' = ush2, 'Usher 3' = ush3)

ushvenn <- ggvenn(ushlist) + scale_fill_manual(values =c("#8A9197FF","#709AE1FF","#FED439FF"))
## Scale for fill is already present.
## Adding another scale for fill, which will replace the existing scale.
ushvenn

 # ggsave("ushvenn.pdf", ushvenn, width = 10, height = 10)


########################################################################################################################
########################################################################################################################
set.seed(1)
Intersect <- function (x) {  
  # Multiple set version of intersect
  # x is a list
  if (length(x) == 1) {
    unlist(x)
  } else if (length(x) == 2) {
    intersect(x[[1]], x[[2]])
  } else if (length(x) > 2){
    intersect(x[[1]], Intersect(x[-1]))
  }
}

Union <- function (x) {  
  # Multiple set version of union
  # x is a list
  if (length(x) == 1) {
    unlist(x)
  } else if (length(x) == 2) {
    union(x[[1]], x[[2]])
  } else if (length(x) > 2) {
    union(x[[1]], Union(x[-1]))
  }
}

Setdiff <- function (x, y) {
  # Remove the union of the y's from the common x's. 
  # x and y are lists of characters.
  xx <- Intersect(x)
  yy <- Union(y)
  setdiff(xx, yy)
}
########################################################################################################################
ushlist <- list(A = ush1,
                B= ush2,
                C= ush3)
shared_ush_differentials <- Intersect(ushlist)

ush1_unique <- Setdiff(ushlist[c("A")], ushlist[c("B","C")])

ush2_unique <- Setdiff(ushlist[c("B")], ushlist[c("A","C")])

ush3_unique <- Setdiff(ushlist[c("C")], ushlist[c("A","B")])

ush12_unique <- Setdiff(ushlist[c("A","B")], ushlist[c("C")])

ush13_unique <- Setdiff(ushlist[c("A","C")], ushlist[c("B")])

ush23_unique <- Setdiff(ushlist[c("B","C")], ushlist[c("A")])
full_derepped_list <- Union(ushlist)

namelist <-c("Common To All","Usher 1 Only","Usher 2 Only", "Usher 3 Only", "Usher 1 and 2", "Usher 1 and 3", "Usher 2 and 3")
miRNAs <- list(shared_ush_differentials,
               ush1_unique,
               ush2_unique,
               ush3_unique,
               ush12_unique,
               ush13_unique,
               ush23_unique)
df <- data.frame(Category = namelist,
                  Total = c(36,20,14,5,2,5,10),
                 miRNAs = c("hsa-miR-222-3p,hsa-miR-424-5p,hsa-miR-503-5p,
hsa-miR-1252-5p,hsa-miR-1246,hsa-miR-182-5p,
hsa-miR-1205,hsa-miR-450a-5p,hsa-miR-654-5p,
hsa-miR-3934-5p,hsa-miR-221-5p,hsa-miR-34a-5p,
hsa-miR-150-5p,hsa-let-7d-5p,hsa-miR-483-3p,
hsa-miR-146b-5p,hsa-miR-5010-3p,hsa-miR-591,
hsa-miR-155-5p,hsa-miR-363-3p,hsa-let-7i-5p,
hsa-miR-194-5p,hsa-miR-28-3p,hsa-miR-10a-5p,
hsa-miR-146a-5p,hsa-miR-337-3p,hsa-miR-200c-3p,
hsa-miR-96-5p,hsa-miR-5196-3p+hsa-miR-6732-3p,hsa-let-7f-5p,
hsa-miR-577,hsa-let-7g-5p,hsa-miR-183-5p,
hsa-let-7b-5p,hsa-miR-148a-3p,hsa-miR-98-5p",
"hsa-miR-151a-5p,hsa-miR-129-2-3p,hsa-miR-519d-3p,hsa-miR-4431,hsa-miR-345-5p,hsa-miR-152-3p,
hsa-miR-1260b,hsa-miR-3916,hsa-miR-4455,hsa-miR-3195,hsa-miR-195-5p,hsa-miR-1226-3p,
hsa-miR-151a-3p,hsa-miR-181b-2-3p,hsa-miR-1287-5p,hsa-miR-324-5p,hsa-miR-484,hsa-miR-16-5p,
hsa-miR-331-3p,hsa-miR-26b-5p",
"hsa-miR-513b-5p,hsa-miR-137,hsa-miR-370-3p,hsa-miR-503-3p,hsa-miR-767-5p,hsa-miR-132-3p,hsa-let-7c-5p,
hsa-miR-1304-5p,hsa-miR-514a-3p,hsa-let-7a-5p,hsa-miR-342-3p,hsa-miR-181a-5p,hsa-miR-181c-5p,hsa-miR-1193
",
"hsa-miR-125a-3p,hsa-miR-494-3p,hsa-miR-125b-5p,hsa-miR-4284,hsa-miR-181a-2-3p",
"hsa-miR-551b-3p,hsa-miR-299-3p",
"hsa-miR-371b-5p,hsa-miR-601,hsa-miR-1827,hsa-miR-27a-3p,hsa-miR-582-5p",
"hsa-miR-574-3p,hsa-miR-223-3p,hsa-miR-1183,hsa-miR-28-5p,hsa-miR-542-5p,hsa-let-7e-5p,hsa-miR-23c,
hsa-miR-221-3p,hsa-miR-374b-5p,hsa-miR-23a-3p"))

#write.table(df, file = "ush1.ush2.ush3.common_miRNAs.txt", sep = "\t")




library(kableExtra)
## 
## Attaching package: 'kableExtra'
## The following object is masked from 'package:dplyr':
## 
##     group_rows
tab<- kbl(df, format = "html", booktabs = T) %>%
  kable_styling(full_width = F, latex_options = c("scale_down")) %>%
  row_spec(0, bold = T) %>%
  column_spec(3, width = "30em")
  #column_spec(1, bold = T, underline = T) %>%
  #column_spec(2, bold = T)
#save_kable(tab, file = "~/Desktop/Usher_miRNA/all_de_mirna_tab.pdf")
tab
Category Total miRNAs
Common To All 36 hsa-miR-222-3p,hsa-miR-424-5p,hsa-miR-503-5p, hsa-miR-1252-5p,hsa-miR-1246,hsa-miR-182-5p, hsa-miR-1205,hsa-miR-450a-5p,hsa-miR-654-5p, hsa-miR-3934-5p,hsa-miR-221-5p,hsa-miR-34a-5p, hsa-miR-150-5p,hsa-let-7d-5p,hsa-miR-483-3p, hsa-miR-146b-5p,hsa-miR-5010-3p,hsa-miR-591, hsa-miR-155-5p,hsa-miR-363-3p,hsa-let-7i-5p, hsa-miR-194-5p,hsa-miR-28-3p,hsa-miR-10a-5p, hsa-miR-146a-5p,hsa-miR-337-3p,hsa-miR-200c-3p, hsa-miR-96-5p,hsa-miR-5196-3p+hsa-miR-6732-3p,hsa-let-7f-5p, hsa-miR-577,hsa-let-7g-5p,hsa-miR-183-5p, hsa-let-7b-5p,hsa-miR-148a-3p,hsa-miR-98-5p
Usher 1 Only 20 hsa-miR-151a-5p,hsa-miR-129-2-3p,hsa-miR-519d-3p,hsa-miR-4431,hsa-miR-345-5p,hsa-miR-152-3p, hsa-miR-1260b,hsa-miR-3916,hsa-miR-4455,hsa-miR-3195,hsa-miR-195-5p,hsa-miR-1226-3p, hsa-miR-151a-3p,hsa-miR-181b-2-3p,hsa-miR-1287-5p,hsa-miR-324-5p,hsa-miR-484,hsa-miR-16-5p, hsa-miR-331-3p,hsa-miR-26b-5p
Usher 2 Only 14 hsa-miR-513b-5p,hsa-miR-137,hsa-miR-370-3p,hsa-miR-503-3p,hsa-miR-767-5p,hsa-miR-132-3p,hsa-let-7c-5p, hsa-miR-1304-5p,hsa-miR-514a-3p,hsa-let-7a-5p,hsa-miR-342-3p,hsa-miR-181a-5p,hsa-miR-181c-5p,hsa-miR-1193
Usher 3 Only 5 hsa-miR-125a-3p,hsa-miR-494-3p,hsa-miR-125b-5p,hsa-miR-4284,hsa-miR-181a-2-3p
Usher 1 and 2 2 hsa-miR-551b-3p,hsa-miR-299-3p
Usher 1 and 3 5 hsa-miR-371b-5p,hsa-miR-601,hsa-miR-1827,hsa-miR-27a-3p,hsa-miR-582-5p
Usher 2 and 3 10 hsa-miR-574-3p,hsa-miR-223-3p,hsa-miR-1183,hsa-miR-28-5p,hsa-miR-542-5p,hsa-let-7e-5p,hsa-miR-23c, hsa-miR-221-3p,hsa-miR-374b-5p,hsa-miR-23a-3p

Heatmap summarizing DE miRNAs

library(ComplexHeatmap)
library(genefilter)
## 
## Attaching package: 'genefilter'
## The following object is masked from 'package:ComplexHeatmap':
## 
##     dist2
## The following objects are masked from 'package:MatrixGenerics':
## 
##     rowSds, rowVars
## The following objects are masked from 'package:matrixStats':
## 
##     rowSds, rowVars
library(circlize)
## ========================================
## circlize version 0.4.14
## CRAN page: https://cran.r-project.org/package=circlize
## Github page: https://github.com/jokergoo/circlize
## Documentation: https://jokergoo.github.io/circlize_book/book/
## 
## If you use it in published research, please cite:
## Gu, Z. circlize implements and enhances circular visualization
##   in R. Bioinformatics 2014.
## 
## This message can be suppressed by:
##   suppressPackageStartupMessages(library(circlize))
## ========================================
library(dendextend)
## Registered S3 method overwritten by 'dendextend':
##   method     from 
##   rev.hclust vegan
## 
## ---------------------
## Welcome to dendextend version 1.15.2
## Type citation('dendextend') for how to cite the package.
## 
## Type browseVignettes(package = 'dendextend') for the package vignette.
## The github page is: https://github.com/talgalili/dendextend/
## 
## Suggestions and bug-reports can be submitted at: https://github.com/talgalili/dendextend/issues
## You may ask questions at stackoverflow, use the r and dendextend tags: 
##   https://stackoverflow.com/questions/tagged/dendextend
## 
##  To suppress this message use:  suppressPackageStartupMessages(library(dendextend))
## ---------------------
## 
## Attaching package: 'dendextend'
## The following object is masked from 'package:permute':
## 
##     shuffle
## The following object is masked from 'package:data.table':
## 
##     set
## The following object is masked from 'package:stats':
## 
##     cutree
library(dendsort)
col_fun = colorRamp2(c(-2, 0, 2), c("#D62728FF", "white", "#2CA02CFF"))
mat <- scale(t(ush_vs_cont_expr_counts)) #scale normalizes count data: z = (x — u) / s; x=count u=col_mean s=stddev
mat_trim <- mat[,colnames(mat) %in% full_derepped_list]

mat_trim_common <- mat[,colnames(mat) %in% shared_ush_differentials]

mat_trim_ush1 <- mat[,colnames(mat) %in% ush1_unique]

mat_trim_ush2 <- mat[,colnames(mat) %in% ush2_unique]

mat_trim_ush3 <- mat[,colnames(mat) %in% ush3_unique]

mat_trim_ush12 <- mat[,colnames(mat) %in% ush12_unique]

mat_trim_ush13 <- mat[,colnames(mat) %in% ush13_unique]

mat_trim_ush23 <- mat[,colnames(mat) %in% ush23_unique]
#Heatmap(scale(t(miRNA_final_count_tab)))#this is the same as below...

row_dend = as.dendrogram(hclust(dist(mat_trim)))
#row_dend = color_branches(row_dend, k = 5,col=c("#8A9197FF","#709AE1FF","#C80813FF","#46732EFF","orange"))
col_dend = dendsort(hclust(dist(t(mat_trim))))
#pdf(file = "final_htmap.pdf", width = 12, height = 8, fonts = "Times")
fhp <- Heatmap(mat_trim, name = "Scale", 
        cluster_rows = row_dend, 
        row_split = 2,
        row_title = c("Control","Usher"),
        row_dend_reorder = TRUE,
        row_title_rot = 0,
        column_km = 6,
        column_title = LETTERS[1:6],
        col= col_fun, 
        column_names_rot = 45,
        column_names_gp = gpar(fontsize=8))
#dev.off()
 

fhp

#common heatmap
common_heatmap <- Heatmap(mat_trim_common, name = "Scale", 
        cluster_rows = row_dend, 
        row_split = 2,
        row_title = c("Control","Usher"),
        row_dend_reorder = TRUE,
        row_title_rot = 0,
        column_km = 6,
        column_title = LETTERS[1:6],
        col= col_fun, 
        column_names_rot = 45,
        column_names_gp = gpar(fontsize=8))
#pdf(file = "common_htmap.pdf", width = 12, height = 8, fonts = "Times")
common_heatmap

#dev.off()

#ush1_only_htmp
ush1_heatmap <- Heatmap(mat_trim_ush1, name = "Scale", 
        cluster_rows = row_dend, 
        row_split = 2,
        row_title = c("Control","Usher"),
        row_dend_reorder = TRUE,
        row_title_rot = 0,
        column_km = 6,
        column_title = LETTERS[1:6],
        col= col_fun, 
        column_names_rot = 45,
        column_names_gp = gpar(fontsize=8))
#pdf(file = "ush1_htmap.pdf", width = 12, height = 8, fonts = "Times")
ush1_heatmap

#dev.off()

#ush2_only_htmp
ush2_heatmap <- Heatmap(mat_trim_ush2, name = "Scale", 
        cluster_rows = row_dend, 
        row_split = 2,
        row_title = c("Control","Usher"),
        row_dend_reorder = TRUE,
        row_title_rot = 0,
        column_km = 6,
        column_title = LETTERS[1:6],
        col= col_fun, 
        column_names_rot = 45,
        column_names_gp = gpar(fontsize=8))
#pdf(file = "ush2_htmap.pdf", width = 12, height = 8, fonts = "Times")
ush2_heatmap

#dev.off()

#ush 3 heatmap only has one miRNA
#ush3_heatmap <- Heatmap(mat_trim_ush3, name = "Scale", 
        #cluster_rows = row_dend, 
        #row_split = 2,
        #row_title = c("Control","Usher"),
        #row_dend_reorder = TRUE,
        #row_title_rot = 0,
        #column_km = 2,
        #column_title = LETTERS[1:2],
        #col= col_fun, 
        #column_namerot = 45,
        #column_namegp = gpar(fontsize=8))
#pdf(file = "ush3_htmap.pdf", width = 12, height = 8, fonts = "Times")
#ush3_heatmap
#dev.off()

#usher1 and 2 only
#ush12_heatmap <- Heatmap(mat_trim_ush12, name = "Scale", 
 #       cluster_rows = row_dend, 
  #      row_split = 2,
   #    row_dend_reorder = TRUE,
    #    row_title_rot = 0,
    #    column_km = 2,
     #   column_title = LETTERS[1:2],
      #  col= col_fun, 
       # column_namerot = 45,
        #column_namegp = gpar(fontsize=8))
#pdf(file = "ush12_htmap.pdf", width = 12, height = 8, fonts = "Times")
#ush12_heatmap
#dev.off()

#usher 1 and 3

#ush13_heatmap <- Heatmap(mat_trim_ush13, name = "Scale", 
 #       cluster_rows = row_dend, 
  #      row_split = 2,
   #     row_title = c("Control","Usher"),
    #    row_dend_reorder = TRUE,
     #   row_title_rot = 0,
      #  column_km = 2,
       # column_title = LETTERS[1:2],
        #col= col_fun, 
        #column_namerot = 45,
        #column_namegp = gpar(fontsize=8))
#pdf(file = "ush13_htmap.pdf", width = 12, height = 8, fonts = "Times")
#ush13_heatmap
#dev.off()


#Usher 2 and 3

#ush23_heatmap <- Heatmap(mat_trim_ush23, name = "Scale", 
 #       cluster_rows = row_dend, 
  #      row_split = 2,
   #     row_title = c("Control","Usher"),
    #    row_dend_reorder = TRUE,
     #   row_title_rot = 0,
      #  column_km = 6,
       # column_title = LETTERS[1:6],
        #col= col_fun, 
        #column_namerot = 45,
        #column_namegp = gpar(fontsize=8))
#pdf(file = "ush23_htmap.pdf", width = 12, height = 8, fonts = "Times")
#ush23_heatmap
#dev.off()

#the top 22 PCA and DESeq consistent

top.miRNAS<- c("hsa-miR-146a-5p","hsa-miR-155-5p","hsa-miR-16-5p","hsa-miR-29b-3p",
"hsa-miR-19b-3p",
"hsa-miR-4454+hsa-miR-7975",
"hsa-miR-142-3p",
"hsa-miR-96-5p",
"hsa-miR-182-5p",
"hsa-miR-183-5p",
"hsa-miR-181b-2-3p",
"hsa-miR-519d-3p",
"hsa-miR-566",
"hsa-miR-4431",
"hsa-miR-484",
"hsa-miR-331-3p",
"hsa-miR-296-5p",
"hsa-miR-15a-5p",
"hsa-miR-129-2-3p",
"hsa-miR-337-3p",
"hsa-miR-223-3p",
"hsa-miR-21-5p")

make some tables of the differential miRNAs

#common_sigtab <- 

ush1_only_sigtab <- ush1.nbt.sigtab2[row.names(ush1.nbt.sigtab2) %in% ush1_unique,]
ush1_only_sigtab <- ush1_only_sigtab[,1:6]

ush2_only_sigtab <- ush2.nbt.sigtab2[row.names(ush2.nbt.sigtab2) %in% ush2_unique,]
ush2_only_sigtab <- ush2_only_sigtab[,1:6]

ush3_only_sigtab <- ush3.nbt.sigtab2[row.names(ush3.nbt.sigtab2) %in% ush3_unique,]
ush3_only_sigtab <- ush3_only_sigtab[,1:6]

#pat.ctrl_only_sigtab <- pat.ctrl.sigtab2[row.names(pat.ctrl.sigtab2) %in% pat.ctrl_unique,]
#pat.ctrl_only_sigtab <- pat.ctrl_only_sigtab[,1:6]

#car.ctrl_only_sigtab <- car.ctrl.sigtab2[row.names(car.ctrl.sigtab2) %in% car.ctrl_unique,]
#car.ctrl_only_sigtab <- car.ctrl_only_sigtab[,1:6]

cvp_mirs <- c("hsa-miR-591","hsa-miR-1226-3p","hsa-miR-513b-5p","hsa-miR-10a-5p","hsa-let-7f-5p","hsa-miR-5196-3p+hsa-miR-6732-3p","hsa-miR-183-5p","hsa-miR-551b-3p","hsa-miR-132-3p","hsa-miR-542-5p","hsa-miR-299-3p","hsa-miR-92b-3p","hsa-miR-517c-3p+hsa-miR-519a-3p","hsa-miR-1291","hsa-miR-874-3p","hsa-miR-362-3p","hsa-miR-889-3p","hsa-miR-142-5p","hsa-miR-338-5p","hsa-miR-518b","hsa-miR-1244","hsa-miR-449b-5p","hsa-miR-450a-2-3p","hsa-miR-1183","hsa-miR-608","hsa-miR-211-5p","hsa-miR-2053","hsa-miR-181c-5p","hsa-miR-95-3p","hsa-miR-492","hsa-miR-296-5p","hsa-miR-324-5p")

mat1 <- t(ush_vs_cont_expr_counts)
mat2 <- mat1[,colnames(mat1) %in% cvp_mirs]
write.table(mat2, file = "patient_carrier_miRNA_differential_count_tab.tsv", sep = "\t")


write.table(ush1.nbt.sigtab2, file = "ush1.nbt.sigtab.tsv", sep = "\t")
write.table(ush2.nbt.sigtab2, file = "ush2.nbt.sigtab.tsv", sep = "\t")
write.table(ush3.nbt.sigtab2, file = "ush3.nbt.sigtab.tsv", sep = "\t")

write.table(ush1_only_sigtab, file = "ush1.only.sigtab.tsv", sep = "\t")
write.table(ush2_only_sigtab, file = "ush2.only.sigtab.tsv", sep = "\t")
write.table(ush3_only_sigtab, file = "ush3.only.sigtab.tsv", sep = "\t")

write.table(shared_ush_differentials, file = "common_miRNAlist.tsv", sep = "\t")

write.table(ush12_unique, file = "ush12_miRNAlist.tsv", sep = "\t")
write.table(ush13_unique, file = "ush13_miRNAlist.tsv", sep = "\t")
write.table(ush23_unique, file = "ush23_miRNAlist.tsv", sep = "\t")

write.table(ush1_unique, file = "ush1_miRNAlist.tsv", sep = "\t")
write.table(ush2_unique, file = "ush2_miRNAlist.tsv", sep = "\t")
write.table(ush3_unique, file = "ush3_miRNAlist.tsv", sep = "\t")

mat1 <- t(ush_vs_cont_expr_counts)
mat3 <- mat1[,colnames(mat1) %in% full_derepped_list]
write.table(mat3, file = "all_miRNA_differential_count_tab.tsv", sep = "\t")
#need to make a subset of the top candidates of differential miRNAs, make a master heatmap, then create a table with logfold changes and adjusted p-values etc.

#the top 12 PCA and DESeq consistent

top.miRNAS<- c("hsa-miR-146a-5p",
"hsa-miR-155-5p",
"hsa-miR-16-5p",
"hsa-miR-29b-3p",
"hsa-miR-19b-3p",
"hsa-miR-4454+hsa-miR-7975",
"hsa-miR-142-3p",
"hsa-miR-96-5p",
"hsa-miR-182-5p",
"hsa-miR-183-5p",
"hsa-miR-484",
"hsa-miR-331-3p",
"hsa-miR-296-5p",
"hsa-miR-15a-5p",
"hsa-miR-150-5p",
"hsa-let-7a-5p",
"hsa-miR-23a-3p",
"hsa-miR-342-3p",
"hsa-miR-4284",
"hsa-miR-26b-5p",
"hsa-miR-223-3p",
"hsa-miR-21-5p")
topmat <- mat[,colnames(mat) %in% top.miRNAS]
View(topmat)
ush_vs_cont_pheno_rename <- ush_vs_cont_pheno
rownames(ush_vs_cont_pheno_rename) <- with(ush_vs_cont_pheno_rename, paste(sampleID,phenotype,genotype, sep = ":"))
View(ush_vs_cont_pheno_rename)
rownames(topmat) <- rownames(ush_vs_cont_pheno_rename)

 
top_heatmap <- Heatmap(topmat, name = "Scale", 
        cluster_rows = T, 
        row_split = 3,
        row_title = c("Control","Usher 2 & 3","Usher 1"),
        row_dend_reorder = TRUE,
        row_title_rot = 0,
        column_km = 2,
        column_title = LETTERS[1:2],
        col= col_fun, 
        column_names_rot = 45,
        column_names_gp = gpar(fontsize=14))
pdf(file = "top_htmap.pdf", width = 12, height = 8, fonts = "Times")
top_heatmap
dev.off()
## quartz_off_screen 
##                 2
#need to make a subset of the top candidates of differential miRNAs, make a master heatmap, then create a table with logfold changes and adjusted p-values etc.

#the 12 miRNAs from ddPCR, and the top PCA miRNAs

top.miRNAS<- c(
"hsa-miR-155-5p",
"hsa-miR-16-5p",
"hsa-miR-19b-3p",
"hsa-miR-4454+hsa-miR-7975",
"hsa-miR-142-3p",
"hsa-miR-96-5p",
"hsa-miR-182-5p",
"hsa-miR-183-5p",
"hsa-miR-363-3p",
"hsa-miR-150-5p",
"hsa-let-7a-5p",
"hsa-miR-28-5p",
"hsa-miR-223-3p")
topmat <- mat[,colnames(mat) %in% top.miRNAS]
View(topmat)
ush_vs_cont_pheno_rename <- ush_vs_cont_pheno
rownames(ush_vs_cont_pheno_rename) <- with(ush_vs_cont_pheno_rename, paste(sampleID,phenotype,genotype, sep = ":"))
View(ush_vs_cont_pheno_rename)
rownames(topmat) <- rownames(ush_vs_cont_pheno_rename)
v <- c("Usher-1D-(rep 1):CDH23","Usher-1D-(rep 2):CDH23","Usher-1D-(rep 3):CDH23","Usher-1B-(rep 1):MYO7A","Usher-1B-(rep 2):MYO7A","Control-(rep 1):Normal","Control-(rep 2):Normal","Control-(rep 3):Normal","Usher-3A-(rep 1):CLRN1","Usher-3A-(rep 2):CLRN1","Usher-3A-(rep 3):CLRN1","Usher-3A-(rep 4):CLRN1","Control-(rep 4):Normal","Usher-2A-(rep 1)::USH2A","Usher-2A-(rep 2)::USH2A","Usher-2A-(rep 3):USH2A","Usher-2A-(rep 4):USH2A")
rownames(topmat) <- v

testrnm <- topmat[c(6:8,13,4:5,1:3,14:17,9:12),] 
top_heatmap <- Heatmap(testrnm, name = "Scale", 
       cluster_rows = T, 
       #row_split = 4,
       #row_title = c("Control","Usher 1","Usher 2","Usher 3"),
       row_dend_reorder = TRUE,
       row_title_rot = 0,
       column_km = 2,
       column_title = LETTERS[1:2],
       col= col_fun,
       column_names_rot = 60,
       column_names_gp = gpar(fontsize=14))
       
#pdf(file = "top_htmap.pdf", width = 12, height = 8, fonts = "Times")
top_heatmap

#dev.off()
top_counts <- ush_vs_cont_expr_counts[row.names(ush_vs_cont_expr_counts) %in% top.miRNAS,]
top_ush1 <- ush1.nbt.sigtab[row.names(ush1.nbt.sigtab) %in% top.miRNAS,1:6]
top_ush2 <- ush2.nbt.sigtab[row.names(ush2.nbt.sigtab) %in% top.miRNAS,1:6]
top_ush3 <-ush3.nbt.sigtab[row.names(ush3.nbt.sigtab) %in% top.miRNAS,1:6]


write.table(top_counts, file = "top_counts.tsv", sep = "\t")
write.table(top_ush1, file = "top_ush1.tsv", sep = "\t")
write.table(top_ush2, file = "top_ush2.tsv", sep = "\t")
write.table(top_ush3, file = "top_ush3.tsv", sep = "\t")
mut_tab <- read.delim("ush_variant_tab.txt")
colnames(mut_tab) <- c("Cell Line","Phenotype","Chromosome Position","Ref/Alt","Genotype","Classification","ACMG Classification Criteria","HGVS cDot","Sequence Ontology","Gene Name","Gene Inheritance","Associated Conditions")

tab2 <- kbl(mut_tab, format = "html", booktabs = T) %>%
  kable_styling(full_width = F, latex_options = c("scale_down")) %>%
  row_spec(0, bold = T) %>%
  column_spec(12, width = "10em") %>%
  column_spec(1, bold = T, underline = T)
tab2
Cell Line Phenotype Chromosome Position Ref/Alt Genotype Classification ACMG Classification Criteria HGVS cDot Sequence Ontology Gene Name Gene Inheritance Associated Conditions
D3739 Usher-1D 10:71779316 G/A Homozygous Likely Pathogenic PM2, PS1, PP3 NM_022124.6:c.5237G>A missense CDH23 Recessive Usher syndrome type 1D, CDH23-Related Disorders, Autosomal recessive nonsyndromic hearing loss 12;Pituitary adenoma 5, Rare genetic deafness, Retinal dystrophy, Childhood onset hearing loss, Usher syndrome
D3741 Usher-1B 11:77181589 G/T Homozygous Likely Pathogenic PM2, PP2, PS1, PP3 NM_000260.4:c.2904G>T missense MYO7A Recessive Rare genetic deafness, Autosomal recessive nonsyndromic hearing loss 2, Usher syndrome type 1B
D2880 Usher-3A 3:150928107 A/C Homozygous VUS/Conflicting BS1, PVS1 Strong, PP5 NM_174878.3:c.528T>G stop gained CLRN1 Recessive Usher syndrome type 3, Rare genetic deafness, Retinitis pigmentosa 61;Usher syndrome type 3A
Coriell(GM09053) Usher-2A 1:216190280 AG/- Heterozygous Pathogenic PM2, PVS1, PP5 NM_206933.4:c.4338_4339delCT frameshift USH2A Recessive Usher syndrome type 2A, USH2A-Related Disorders, Retinal dystrophy, Retinitis pigmentosa 39;Usher syndrome type 2A
1:215647526 T/- Heterozygous Pathogenic PM2, PVS1, PP5 NM_206933.4:c.14787delA frameshift USH2A Recessive Retinal dystrophy

ddPCR results

Read in data

library(tidyverse)
## ── Attaching packages ─────────────────────────────────────── tidyverse 1.3.1 ──
## ✔ tibble  3.2.1     ✔ purrr   1.0.1
## ✔ tidyr   1.3.0     ✔ forcats 0.5.1
## ✔ readr   2.1.2
## ── Conflicts ────────────────────────────────────────── tidyverse_conflicts() ──
## ✖ dplyr::between()         masks data.table::between()
## ✖ IRanges::collapse()      masks dplyr::collapse()
## ✖ Biobase::combine()       masks BiocGenerics::combine(), dplyr::combine(), gridExtra::combine()
## ✖ matrixStats::count()     masks dplyr::count()
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## ✖ purrr::transpose()       masks data.table::transpose()
library(ggpubr)
## 
## Attaching package: 'ggpubr'
## The following object is masked from 'package:dendextend':
## 
##     rotate
## The following object is masked from 'package:microViz':
## 
##     stat_chull
library(rstatix)
## 
## Attaching package: 'rstatix'
## The following object is masked from 'package:genefilter':
## 
##     Anova
## The following object is masked from 'package:IRanges':
## 
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##     filter
miRNA_ddpcr_down <- read.delim("ddpcr_down.txt", stringsAsFactors=TRUE)

miRNA_ddpcr_up <- read.delim("ddpcr_up.txt", stringsAsFactors=TRUE)

levels(miRNA_ddpcr_down$Sample) <- c("CONTROL","USH1B","USH1D","USH2A","USH3A")
levels(miRNA_ddpcr_down$Sample)
## [1] "CONTROL" "USH1B"   "USH1D"   "USH2A"   "USH3A"
levels(miRNA_ddpcr_up$Sample) <- c("CONTROL","USH1B","USH1D","USH2A","USH3A")
levels(miRNA_ddpcr_up$Sample)
## [1] "CONTROL" "USH1B"   "USH1D"   "USH2A"   "USH3A"

ANOVAs with Tukey pairwise comparisons:

363-3p

library(dplyr)
library(ggplot2)
library(multcompView)
library(ggthemes)
library(ggsci)


data_summary <- function(data, varname, groupnames){
  require(plyr)
  summary_func <- function(x, col){
    c(mean = mean(x[[col]], na.rm=TRUE),
      sd = sd(x[[col]], na.rm=TRUE))
  }
  data_sum<-ddply(data, groupnames, .fun=summary_func,
                  varname)
  data_sum <- rename(data_sum, c("mean" = varname))
 return(data_sum)
}

# Compute the analysis of variance
res.aov363 <- aov( miRNA_363_3p ~ Sample, data = miRNA_ddpcr_up)
# Summary of the analysis
sum_aov_363 <- data_summary(miRNA_ddpcr_up,varname = "miRNA_363_3p", groupnames = "Sample")
## Loading required package: plyr
## ------------------------------------------------------------------------------
## You have loaded plyr after dplyr - this is likely to cause problems.
## If you need functions from both plyr and dplyr, please load plyr first, then dplyr:
## library(plyr); library(dplyr)
## ------------------------------------------------------------------------------
## 
## Attaching package: 'plyr'
## The following objects are masked from 'package:rstatix':
## 
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## 
##     arrange, count, desc, failwith, id, mutate, rename, summarise,
##     summarize
tukey363 <- TukeyHSD(res.aov363)
summary(res.aov363)
##             Df Sum Sq Mean Sq F value Pr(>F)    
## Sample       4 594204  148551   146.1 <2e-16 ***
## Residuals   51  51842    1017                   
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering363 <- multcompLetters4(res.aov363,tukey363) #
group_lettering363
## $Sample
##   USH1D   USH2A   USH1B   USH3A CONTROL 
##     "a"     "b"     "b"     "c"     "d"
group_lettering363 <- data.frame(group_lettering363$Sample$Letters)
group_lettering363$Sample <- rownames(group_lettering363)
group_lettering363$Sample <- c("USH1D","USH2A","USH1B","USH3A","CONTROL")
sum_aov_363 <- merge(sum_aov_363,group_lettering363, by = "Sample" )
colnames(sum_aov_363)[4] <- "group_lettering"

p363 <- ggplot(sum_aov_363, aes(x=reorder(Sample,+miRNA_363_3p), y=miRNA_363_3p, fill=Sample)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(miRNA_363_3p-sd,0), ymax=miRNA_363_3p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = miRNA_363_3p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-363-3p ") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-363-3p copies/ng RNA")+
  scale_x_discrete(guide = guide_axis(angle = 45))

p363

#ggsave("USH_miR-155-5p_anova.pdf", plot = p1, device = 'pdf', width = 7, height = 7)

150-5p

# Compute the analysis of variance
res.aov150 <- aov( miRNA_150_5p ~ Sample, data = miRNA_ddpcr_up)
# Summary of the analysis
sum_aov_150 <- data_summary(miRNA_ddpcr_up,varname = "miRNA_150_5p", groupnames = "Sample")
tukey150 <- TukeyHSD(res.aov150)
summary(res.aov150)
##             Df   Sum Sq Mean Sq F value Pr(>F)    
## Sample       4 17895511 4473878   365.7 <2e-16 ***
## Residuals   51   623914   12234                   
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering150 <- multcompLetters4(res.aov150,tukey150) #
group_lettering150
## $Sample
##   USH2A   USH3A   USH1D   USH1B CONTROL 
##     "a"     "b"     "b"     "b"     "b"
group_lettering150 <- data.frame(group_lettering150$Sample$Letters)
group_lettering150$Sample <- rownames(group_lettering150)
sum_aov_150 <- merge(sum_aov_150,group_lettering150, by = "Sample" )
colnames(sum_aov_150)[4] <- "group_lettering"

p150 <- ggplot(sum_aov_150, aes(x=reorder(Sample,+miRNA_150_5p), y=miRNA_150_5p, fill=Sample)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(miRNA_150_5p-sd,0), ymax=miRNA_150_5p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = miRNA_150_5p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-150-5p ") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-150-5p copies/ng RNA")+
  scale_x_discrete(guide = guide_axis(angle = 45))
p150

#ggsave("USH_miR-155-5p_anova.pdf", plot = p1, device = 'pdf', width = 7, height = 7)

155-5p

# Compute the analysis of variance
res.aov155 <- aov( miRNA_155_5p ~ Sample, data = miRNA_ddpcr_up)
# Summary of the analysis
sum_aov_155 <- data_summary(miRNA_ddpcr_up,varname = "miRNA_155_5p", groupnames = "Sample")
tukey155 <- TukeyHSD(res.aov155)
summary(res.aov155)
##             Df    Sum Sq   Mean Sq F value Pr(>F)    
## Sample       4 6.954e+10 1.739e+10   493.8 <2e-16 ***
## Residuals   51 1.796e+09 3.521e+07                   
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering155 <- multcompLetters4(res.aov155,tukey155) #
group_lettering155
## $Sample
##   USH2A   USH1B   USH1D   USH3A CONTROL 
##     "a"     "b"     "b"     "c"     "d"
group_lettering155 <- data.frame(group_lettering155$Sample$Letters)
group_lettering155$Sample <- rownames(group_lettering155)
sum_aov_155 <- merge(sum_aov_155,group_lettering155, by = "Sample" )
colnames(sum_aov_155)[4] <- "group_lettering"

p155 <- ggplot(sum_aov_155, aes(x=reorder(Sample,+miRNA_155_5p), y=miRNA_155_5p, fill=Sample)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(miRNA_155_5p-sd,0), ymax=miRNA_155_5p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = miRNA_155_5p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-155-5p ") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-155-5p copies/ng RNA")+
  scale_x_discrete(guide = guide_axis(angle = 45))

p155

#ggsave("USH_miR-155-5p_anova.pdf", plot = p1, device = 'pdf', width = 7, height = 7)

223-3p

# Compute the analysis of variance
res.aov223 <- aov( miRNA_223_3p ~ Sample, data = miRNA_ddpcr_up)
# Summary of the analysis
sum_aov_223 <- data_summary(miRNA_ddpcr_up,varname = "miRNA_223_3p", groupnames = "Sample")
tukey223 <- TukeyHSD(res.aov223)
summary(res.aov223)
##             Df  Sum Sq Mean Sq F value Pr(>F)    
## Sample       4 4974630 1243658   168.2 <2e-16 ***
## Residuals   51  377032    7393                   
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering223 <- multcompLetters4(res.aov223,tukey223) #
group_lettering223
## $Sample
##   USH2A   USH1D   USH3A CONTROL   USH1B 
##     "a"     "b"     "b"     "c"     "c"
group_lettering223 <- data.frame(group_lettering223$Sample$Letters)
group_lettering223$Sample <- rownames(group_lettering223)
sum_aov_223 <- merge(sum_aov_223,group_lettering223, by = "Sample" )
colnames(sum_aov_223)[4] <- "group_lettering"

p223 <- ggplot(sum_aov_223, aes(x=reorder(Sample,+miRNA_223_3p), y=miRNA_223_3p, fill=Sample)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(miRNA_223_3p-sd,0), ymax=miRNA_223_3p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = miRNA_223_3p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-223-3p ") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-223-3p copies/ng RNA")+
  scale_x_discrete(guide = guide_axis(angle = 45))
  

p223

#ggsave("USH_miR-155-3p_anova.pdf", plot = p1, device = 'pdf', width = 7, height = 7)

7a-5p

# Compute the analysis of variance
res.aov7a <- aov( let_7a_5p ~ Sample, data = miRNA_ddpcr_up)
# Summary of the analysis
sum_aov_7a <- data_summary(miRNA_ddpcr_up,varname = "let_7a_5p", groupnames = "Sample")
tukey7a <- TukeyHSD(res.aov7a)
summary(res.aov7a)
##             Df    Sum Sq   Mean Sq F value Pr(>F)    
## Sample       4 435962143 108990536   248.9 <2e-16 ***
## Residuals   51  22330312    437849                   
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering7a <- multcompLetters4(res.aov7a,tukey7a) #
group_lettering7a
## $Sample
##   USH2A   USH3A   USH1D   USH1B CONTROL 
##     "a"     "b"     "b"     "c"     "d"
group_lettering7a <- data.frame(group_lettering7a$Sample$Letters)
group_lettering7a$Sample <- rownames(group_lettering7a)
sum_aov_7a <- merge(sum_aov_7a,group_lettering7a, by = "Sample" )
colnames(sum_aov_7a)[4] <- "group_lettering"

p7a <- ggplot(sum_aov_7a, aes(x=reorder(Sample,+let_7a_5p), y=let_7a_5p, fill=Sample)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(let_7a_5p-sd,0), ymax=let_7a_5p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = let_7a_5p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "let-7a-5p ") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("let-7a-5p copies/ng RNA")+
  scale_x_discrete(guide = guide_axis(angle = 45))
p7a

#ggsave("USH_miR-155-5p_anova.pdf", plot = p1, device = 'pdf', width = 7, height = 7)

142-3p

# Compute the analysis of variance
res.aov142 <- aov( miRNA_142_3p ~ Sample, data = miRNA_ddpcr_up)
# Summary of the analysis
sum_aov_142 <- data_summary(miRNA_ddpcr_up,varname = "miRNA_142_3p", groupnames = "Sample")
tukey142 <- TukeyHSD(res.aov142)
summary(res.aov142)
##             Df    Sum Sq   Mean Sq F value Pr(>F)    
## Sample       4 2.083e+11 5.209e+10   206.9 <2e-16 ***
## Residuals   51 1.284e+10 2.517e+08                   
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering142 <- multcompLetters4(res.aov142,tukey142) #
group_lettering142
## $Sample
##   USH2A   USH1B   USH3A CONTROL   USH1D 
##     "a"     "b"     "b"     "c"     "d"
group_lettering142 <- data.frame(group_lettering142$Sample$Letters)
group_lettering142$Sample <- rownames(group_lettering142)
sum_aov_142 <- merge(sum_aov_142,group_lettering142, by = "Sample" )
colnames(sum_aov_142)[4] <- "group_lettering"

p142 <- ggplot(sum_aov_142, aes(x=reorder(Sample,+miRNA_142_3p), y=miRNA_142_3p, fill=Sample)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(miRNA_142_3p-sd,0), ymax=miRNA_142_3p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = miRNA_142_3p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-142-3p ") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-142-3p copies/ng RNA")+
  scale_x_discrete(guide = guide_axis(angle = 45))
  

p142

#ggsave("USH_miR-155-3p_anova.pdf", plot = p1, device = 'pdf', width = 7, height = 7)
library(ggpubr)
total_anovas <- ggarrange(p363,p223,p150,p155,p7a,p142,
          nrow = 2,
          ncol = 3,
          legend = "none",
          labels = "AUTO")
total_anovas

#ggsave(total_anovas, filename = "ddpcr_up.pdf", device = "pdf", width = 8, height = 8)

Downregulated ddPCR miRNAs

96-5p

# Compute the analysis of variance
res.aov96 <- aov( miRNA_96_5p ~ Sample, data = miRNA_ddpcr_down)
# Summary of the analysis
sum_aov_96 <- data_summary(miRNA_ddpcr_down,varname = "miRNA_96_5p", groupnames = "Sample")
tukey96 <- TukeyHSD(res.aov96)
summary(res.aov96)
##             Df Sum Sq Mean Sq F value Pr(>F)    
## Sample       4 146674   36668   484.7 <2e-16 ***
## Residuals   35   2648      76                   
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering96 <- multcompLetters4(res.aov96,tukey96) #
group_lettering96
## $Sample
## CONTROL   USH3A   USH2A   USH1B   USH1D 
##     "a"     "b"     "b"     "b"     "b"
group_lettering96 <- data.frame(group_lettering96$Sample$Letters)
group_lettering96$Sample <- rownames(group_lettering96)
sum_aov_96 <- merge(sum_aov_96,group_lettering96, by = "Sample" )
colnames(sum_aov_96)[4] <- "group_lettering"

p96 <- ggplot(sum_aov_96, aes(x=reorder(Sample,-miRNA_96_5p), y=miRNA_96_5p, fill=Sample)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(miRNA_96_5p-sd,0), ymax=miRNA_96_5p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = miRNA_96_5p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-96-5p ") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-96-5p copies/ng RNA")+
  scale_x_discrete(guide = guide_axis(angle = 45))
  

p96

#ggsave("USH_miR-155-5p_anova.pdf", plot = p1, device = 'pdf', width = 7, height = 7)

182-3p

# Compute the analysis of variance
res.aov182 <- aov( miRNA_182_3p ~ Sample, data = miRNA_ddpcr_down)
# Summary of the analysis
sum_aov_182 <- data_summary(miRNA_ddpcr_down,varname = "miRNA_182_3p", groupnames = "Sample")
tukey182 <- TukeyHSD(res.aov182)
summary(res.aov182)
##             Df  Sum Sq Mean Sq F value Pr(>F)    
## Sample       4 5545167 1386292   799.5 <2e-16 ***
## Residuals   35   60685    1734                   
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering182 <- multcompLetters4(res.aov182,tukey182) #
group_lettering182
## $Sample
## CONTROL   USH3A   USH2A   USH1B   USH1D 
##     "a"     "b"     "b"     "b"     "b"
group_lettering182 <- data.frame(group_lettering182$Sample$Letters)
group_lettering182$Sample <- rownames(group_lettering182)
sum_aov_182 <- merge(sum_aov_182,group_lettering182, by = "Sample" )
colnames(sum_aov_182)[4] <- "group_lettering"

p182 <- ggplot(sum_aov_182, aes(x=reorder(Sample,-miRNA_182_3p), y=miRNA_182_3p, fill=Sample)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(miRNA_182_3p-sd,0), ymax=miRNA_182_3p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = miRNA_182_3p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-182-3p ") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-182-3p copies/ng RNA")+
  scale_x_discrete(guide = guide_axis(angle = 45))
  

p182

#ggsave("USH_miR-155-3p_anova.pdf", plot = p1, device = 'pdf', width = 7, height = 7)

183-3p

# Compute the analysis of variance
res.aov183 <- aov( miRNA_183_3p ~ Sample, data = miRNA_ddpcr_down)
# Summary of the analysis
sum_aov_183 <- data_summary(miRNA_ddpcr_down,varname = "miRNA_183_3p", groupnames = "Sample")
tukey183 <- TukeyHSD(res.aov183)
summary(res.aov183)
##             Df  Sum Sq Mean Sq F value Pr(>F)    
## Sample       4 1177002  294251    1081 <2e-16 ***
## Residuals   35    9529     272                   
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering183 <- multcompLetters4(res.aov183,tukey183) #
group_lettering183
## $Sample
## CONTROL   USH3A   USH2A   USH1B   USH1D 
##     "a"     "b"     "b"     "b"     "b"
group_lettering183 <- data.frame(group_lettering183$Sample$Letters)
group_lettering183$Sample <- rownames(group_lettering183)
sum_aov_183 <- merge(sum_aov_183,group_lettering183, by = "Sample" )
colnames(sum_aov_183)[4] <- "group_lettering"

p183 <- ggplot(sum_aov_183, aes(x=reorder(Sample,-miRNA_183_3p), y=miRNA_183_3p, fill=Sample)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(miRNA_183_3p-sd,0), ymax=miRNA_183_3p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = miRNA_183_3p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-183-3p ") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-183-3p copies/ng RNA")+
  scale_x_discrete(guide = guide_axis(angle = 45))
  

p183

#ggsave("USH_miR-155-3p_anova.pdf", plot = p1, device = 'pdf', width = 7, height = 7)

28-5p

# Compute the analysis of variance
res.aov28 <- aov( miRNA_28_5p ~ Sample, data = miRNA_ddpcr_down)
# Summary of the analysis
sum_aov_28 <- data_summary(miRNA_ddpcr_down,varname = "miRNA_28_5p", groupnames = "Sample")
tukey28 <- TukeyHSD(res.aov28)
summary(res.aov28)
##             Df Sum Sq Mean Sq F value Pr(>F)    
## Sample       4 149553   37388   97.11 <2e-16 ***
## Residuals   35  13475     385                   
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering28 <- multcompLetters4(res.aov28,tukey28) #
group_lettering28
## $Sample
## CONTROL   USH3A   USH1B   USH2A   USH1D 
##     "a"     "b"     "b"     "b"     "b"
group_lettering28 <- data.frame(group_lettering28$Sample$Letters)
group_lettering28$Sample <- rownames(group_lettering28)
sum_aov_28 <- merge(sum_aov_28,group_lettering28, by = "Sample" )
colnames(sum_aov_28)[4] <- "group_lettering"

p28 <- ggplot(sum_aov_28, aes(x=reorder(Sample, -miRNA_28_5p), y=miRNA_28_5p, fill=Sample)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(miRNA_28_5p-sd,0), ymax=miRNA_28_5p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = miRNA_28_5p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-28-5p ") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-28-5p copies/ng RNA")+
  scale_x_discrete(guide = guide_axis(angle = 45))
  

p28

#ggsave("USH_miR-155-5p_anova.pdf", plot = p1, device = 'pdf', width = 7, height = 7)

16-5p

# Compute the analysis of variance
res.aov16 <- aov( miRNA_16_5p ~ Sample, data = miRNA_ddpcr_down)
# Summary of the analysis
sum_aov_16 <- data_summary(miRNA_ddpcr_down,varname = "miRNA_16_5p", groupnames = "Sample")
tukey16 <- TukeyHSD(res.aov16)
summary(res.aov16)
##             Df    Sum Sq   Mean Sq F value Pr(>F)    
## Sample       4 3.844e+09 960920246    1287 <2e-16 ***
## Residuals   35 2.612e+07    746412                   
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering16 <- multcompLetters4(res.aov16,tukey16) #
group_lettering16
## $Sample
## CONTROL   USH2A   USH1B   USH3A   USH1D 
##     "a"     "b"     "c"     "d"     "e"
group_lettering16 <- data.frame(group_lettering16$Sample$Letters)
group_lettering16$Sample <- rownames(group_lettering16)
sum_aov_16 <- merge(sum_aov_16,group_lettering16, by = "Sample" )
colnames(sum_aov_16)[4] <- "group_lettering"

p16 <- ggplot(sum_aov_16, aes(x=reorder(Sample, -miRNA_16_5p), y=miRNA_16_5p, fill=Sample)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(miRNA_16_5p-sd,0), ymax=miRNA_16_5p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = miRNA_16_5p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-16-5p ") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-16-5p copies/ng RNA")+
  scale_x_discrete(guide = guide_axis(angle = 45))
  

p16

#ggsave("USH_miR-155-5p_anova.pdf", plot = p1, device = 'pdf', width = 7, height = 7)

19b-3p

# Compute the analysis of variance
res.aov19b <- aov( miRNA_19b_3p ~ Sample, data = miRNA_ddpcr_down)
# Summary of the analysis
sum_aov_19b <- data_summary(miRNA_ddpcr_down,varname = "miRNA_19b_3p", groupnames = "Sample")
tukey19b <- TukeyHSD(res.aov19b)
summary(res.aov19b)
##             Df    Sum Sq   Mean Sq F value Pr(>F)    
## Sample       4 2.427e+10 6.068e+09    1619 <2e-16 ***
## Residuals   35 1.312e+08 3.748e+06                   
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering19b <- multcompLetters4(res.aov19b,tukey19b) #
group_lettering19b
## $Sample
## CONTROL   USH2A   USH1B   USH3A   USH1D 
##     "a"     "b"     "c"     "d"     "e"
group_lettering19b <- data.frame(group_lettering19b$Sample$Letters)
group_lettering19b$Sample <- rownames(group_lettering19b)
sum_aov_19b <- merge(sum_aov_19b,group_lettering19b, by = "Sample" )
colnames(sum_aov_19b)[4] <- "group_lettering"

p19b <- ggplot(sum_aov_19b, aes(x=reorder(Sample,-miRNA_19b_3p), y=miRNA_19b_3p, fill=Sample)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(miRNA_19b_3p-sd,0), ymax=miRNA_19b_3p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = miRNA_19b_3p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-19b-3p ") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-19b-3p copies/ng RNA")+
  scale_x_discrete(guide = guide_axis(angle = 45))
  

p19b

#ggsave("USH_miR-155-3p_anova.pdf", plot = p1, device = 'pdf', width = 7, height = 7)

combined

down_anovas <- ggarrange(p28,p96,p182,p183,p16,p19b,
          nrow = 2,
          ncol = 3,
          legend = "none",
          labels = "AUTO")
down_anovas

#ggsave(down_anovas, filename = "ddpcr_down.pdf", device = "pdf", width = 8, height = 8)
library(ampvis2)
#uvc.amp <- phyloseq_to_ampvis2(uvc.ps) ##outdated
uvc.amp <- amp_load(uvc.ps)
## Phyloseq object provided. Ignoring anything provided for the metadata, taxonomy, fasta, or tree arguments, using those from the phyloseq object instead.
## Warning: Could not find a column named OTU/ASV in otutable, using rownames as
## OTU ID's
## Warning: Could not find a column named OTU/ASV in taxonomy, using rownames as
## OTU ID's
## 116 OTUs with 0 total abundance across all samples have been removed.
prop_mirna_abs <- amp_heatmap(data = uvc.amp, tax_aggregate = "Species", group_by = "genotype", tax_show = 20)


bar_plot <- uvc.ps %>%
  tax_fix(min_length = 0,
          unknowns = NA,
          anon_unique = T) %>%
  comp_barplot(
    tax_level = "Species", n_taxa = 20, other_name = "Other",
    taxon_renamer = function(x) stringr::str_remove(x, " [ae]t rel."),
    palette = distinct_palette(n = 20, add = "grey90"),
    merge_other = FALSE, bar_outline_colour = "darkgrey"
  )
## Registered S3 method overwritten by 'seriation':
##   method         from 
##   reorder.hclust vegan
bar_plot

#ggsave(prop_mirna_abs, filename = "mirna_props.pdf", device = "pdf", width = 10, height = 10)

#ggsave(bar_plot, filename = "mirna_bars.pdf", device = "pdf", width = 10, height = 10)
fc_tab <- read.delim("fc_tab.txt")

colnames(fc_tab) <- c("microRNA","log2FC","P-Value (BH-adj.)","log2FC","P-Value (BH-adj.)","log2FC", "P-Value (BH-adj.)","Average log2FC","Standard Dev.")
#library(kableExtra)

fctab<- kbl(fc_tab, format = "html", booktabs = T) %>%
  kable_styling(full_width = F, latex_options = c("scale_down")) %>%
  add_header_above(c(" ", "Usher 1" = 2, "Usher 2" = 2, "Usher 3" = 2,"Mean Log2FC vs. Control"=2), bold = T) %>%
  row_spec(0, bold = T) %>%
  #column_spec(3, width = "30em") #%>%
  column_spec(1, bold = T) #%>%
  #column_spec(2, bold = T)
fctab
Usher 1
Usher 2
Usher 3
Mean Log2FC vs. Control
microRNA log2FC P-Value (BH-adj.) log2FC P-Value (BH-adj.) log2FC P-Value (BH-adj.) Average log2FC Standard Dev.
hsa-miR-150-5p 4.8901 0.0000 9.7786 0.0000 6.5893 0.0000 7.0860 2.4818
hsa-miR-155-5p 4.1500 0.0000 4.6412 0.0000 4.6898 0.0000 4.4936 0.2986
hsa-miR-363-3p 4.1282 0.0000 3.3498 0.0000 3.3819 0.0000 3.6200 0.4404
hsa-miR-223-3p 1.6878 0.0002 4.8040 0.0000 3.2343 0.0000 3.2420 1.5581
hsa-let-7a-5p 0.9413 0.0095 2.0768 0.0000 1.9006 0.0000 1.6396 0.6111
hsa-miR-142-3p 0.5645 0.0259 1.6779 0.0000 1.5316 0.0000 1.2580 0.6051
hsa-miR-19b-3p -1.4389 0.0001 -0.9554 0.0293 -1.1786 0.0041 -1.1910 0.2419
hsa-miR-28-5p -1.9993 0.0000 -2.5731 0.0000 -2.0932 0.0000 -2.2219 0.3078
hsa-miR-16-5p -2.1845 0.0000 -1.0125 0.0487 -1.3422 0.0044 -1.5130 0.6044
hsa-miR-183-5p -2.2771 0.0000 -2.6795 0.0000 -2.3467 0.0000 -2.4344 0.2151
hsa-miR-96-5p -2.9386 0.0000 -3.2701 0.0000 -2.7408 0.0000 -2.9832 0.2674
hsa-miR-182-5p -6.2691 0.0024 -8.0376 0.0005 -6.1735 0.0073 -6.8267 1.0498
#save_kable(fctab, file = "mirna_fc.pdf")

ddpcr_fc_tab <- read.delim("ddPCR_log2FC_tab.txt", row.names = 1)
colnames(ddpcr_fc_tab) <- c("log2FC","P-value (Tukey)", "log2FC","P-value (Tukey)", "log2FC","P-value (Tukey)", "log2FC","P-value (Tukey)", "Average log2FC" ,"Standard Dev.")


ddpcr_fc_tab <- kbl(ddpcr_fc_tab, format = "html", booktabs = T) %>%
  kable_styling(full_width = F, latex_options = c("scale_down")) %>%
  add_header_above(c(" ", "USH1B" = 2,"USH1D"=2,"USH2A"=2,"USH3A"=2, "Summary" = 2), bold = T) %>%
  row_spec(0, bold = T) %>%
  #column_spec(4, width = "30em") %>%
  column_spec(1, bold = T) #%>%
  #column_spec(2, bold = T)
ddpcr_fc_tab
USH1B
USH1D
USH2A
USH3A
Summary
log2FC P-value (Tukey) log2FC P-value (Tukey) log2FC P-value (Tukey) log2FC P-value (Tukey) Average log2FC Standard Dev.
miRNA-96-5p -7.8315 0.0000 -8.4164 0.0000 -6.4778 0 -4.4392 0.0000 -6.7912 1.7658
miRNA-182-3p -7.0483 0.0000 -9.4163 0.0000 -6.2993 0 -4.1755 0.0000 -6.7348 2.1624
miRNA-183-3p -6.4993 0.0000 -8.6036 0.0000 -5.7894 0 -4.2602 0.0000 -6.2881 1.8044
miRNA-28-5p -3.7054 0.0000 -4.3792 0.0000 -4.0710 0 -3.5236 0.0000 -3.9198 0.3816
miRNA-19b-3p -1.8154 0.0000 -2.4705 0.0000 -1.0759 0 -2.1000 0.0000 -1.8655 0.5907
miRNA-16-5p -1.6082 0.0000 -2.1104 0.0000 -0.6188 0 -1.8684 0.0000 -1.5514 0.6547
miRNA-223-3p -0.0316 1.0000 1.0612 0.0013 2.9325 0 1.0534 0.0015 1.2539 1.2312
let-7a-5p 0.4492 0.0001 0.8932 0.0000 1.7187 0 1.0197 0.0000 1.0202 0.5260
miRNA-142-3p 0.5956 0.0000 -0.3789 0.0444 1.6618 0 0.5381 0.0000 0.6042 0.8345
miRNA-150-5p 2.7182 0.8725 2.8787 0.8105 7.7341 0 3.5134 0.3914 4.2111 2.3736
miRNA-155-5p 4.3887 0.0000 4.3706 0.0000 4.9500 0 4.0281 0.0000 4.4343 0.3817
miRNA-363-3p 4.5878 0.0000 5.0891 0.0000 4.7358 0 3.4480 0.0000 4.4652 0.7100
#save_kable(ddpcr_fc_tab, file = "~/Desktop/ddpcr_fc_tab.pdf")

Microarray ANOVAS with tukey adjustments for multiple comparisons

make a data tabel of the 12 miRNAs of interest

microarray_counts <- ush_vs_cont_expr_counts[rownames(ush_vs_cont_expr_counts) %in% c("hsa-miR-155-5p","hsa-miR-150-5p","hsa-miR-363-3p","hsa-miR-223-3p","hsa-miR-28-5p","hsa-miR-182-5p","hsa-miR-183-5p","hsa-miR-96-5p","hsa-let-7a-5p","hsa-miR-142-3p","hsa-miR-16-5p","hsa-miR-19b-3p"), ]
microarray_counts <- data.frame(t(microarray_counts))
microarray_counts$sampleID <- rownames(microarray_counts)


newdf <- merge(microarray_counts, ush_vs_cont_pheno_rename, by = "sampleID")
newdf$genotype <- as.factor(newdf$genotype)
levels(newdf$genotype) <- c("USH1D","USH3A","USH1B","CONTROL","USH2A")
levels(newdf$genotype)
## [1] "USH1D"   "USH3A"   "USH1B"   "CONTROL" "USH2A"

28-5p Microarray

# Compute the analysis of variance
res.aov28.ma <- aov( hsa.miR.28.5p ~ genotype, data = newdf)
# Summary of the analysis
sum_aov_28.ma <- data_summary(newdf,varname = "hsa.miR.28.5p", groupnames = "genotype")
tukey28.ma <- TukeyHSD(res.aov28.ma)
summary(res.aov28.ma)
##             Df Sum Sq Mean Sq F value  Pr(>F)   
## genotype     4  41882   10471   6.761 0.00435 **
## Residuals   12  18584    1549                   
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering28.ma <- multcompLetters4(res.aov28.ma,tukey28.ma) #
group_lettering28.ma
## $genotype
## CONTROL   USH1D   USH1B   USH3A   USH2A 
##     "a"     "b"     "b"     "b"     "b"
group_lettering28.ma <- data.frame(group_lettering28.ma$genotype$Letters)
group_lettering28.ma$genotype <- rownames(group_lettering28.ma)
sum_aov_28.ma <- merge(sum_aov_28.ma,group_lettering28.ma, by = "genotype" )
colnames(sum_aov_28.ma)[4] <- "group_lettering"

p28.ma <- ggplot(sum_aov_28.ma, aes(x=reorder(genotype,-hsa.miR.28.5p), y=hsa.miR.28.5p, fill=genotype)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(hsa.miR.28.5p-sd,0), ymax=hsa.miR.28.5p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = hsa.miR.28.5p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-28-5p") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-28-5p Count")
  

p28.ma

19b-3p Microarray

# Compute the analysis of variance
res.aov19b.ma <- aov( hsa.miR.19b.3p ~ genotype, data = newdf)
# Summary of the analysis
sum_aov_19b.ma <- data_summary(newdf,varname = "hsa.miR.19b.3p", groupnames = "genotype")
tukey19b.ma <- TukeyHSD(res.aov19b.ma)
summary(res.aov19b.ma)
##             Df    Sum Sq  Mean Sq F value     Pr(>F)    
## genotype     4 100990175 25247544   28.81 0.00000453 ***
## Residuals   12  10516459   876372                       
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering19b.ma <- multcompLetters4(res.aov19b.ma,tukey19b.ma) #
group_lettering19b.ma
## $genotype
## CONTROL   USH1B   USH2A   USH3A   USH1D 
##     "a"     "b"    "bc"    "bc"     "c"
group_lettering19b.ma <- data.frame(group_lettering19b.ma$genotype$Letters)
group_lettering19b.ma$genotype <- rownames(group_lettering19b.ma)
sum_aov_19b.ma <- merge(sum_aov_19b.ma,group_lettering19b.ma, by = "genotype" )
colnames(sum_aov_19b.ma)[4] <- "group_lettering"

p19b.ma <- ggplot(sum_aov_19b.ma, aes(x=reorder(genotype,-hsa.miR.19b.3p), y=hsa.miR.19b.3p, fill=genotype)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(hsa.miR.19b.3p-sd,0), ymax=hsa.miR.19b.3p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = hsa.miR.19b.3p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-19b-3p ") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-19b-3p Count")
  

p19b.ma

16-5p Microarray

# Compute the analysis of variance
res.aov16.ma <- aov( hsa.miR.16.5p ~ genotype, data = newdf)
# Summary of the analysis
sum_aov_16.ma <- data_summary(newdf,varname = "hsa.miR.16.5p", groupnames = "genotype")
tukey16.ma <- TukeyHSD(res.aov16.ma)
summary(res.aov16.ma)
##             Df    Sum Sq   Mean Sq F value      Pr(>F)    
## genotype     4 560595898 140148975   49.68 0.000000227 ***
## Residuals   12  33851934   2820994                        
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering16.ma <- multcompLetters4(res.aov16.ma,tukey16.ma) #
group_lettering16.ma
## $genotype
## CONTROL   USH2A   USH3A   USH1B   USH1D 
##     "a"     "b"     "b"    "bc"     "c"
group_lettering16.ma <- data.frame(group_lettering16.ma$genotype$Letters)
group_lettering16.ma$genotype <- rownames(group_lettering16.ma)
sum_aov_16.ma <- merge(sum_aov_16.ma,group_lettering16.ma, by = "genotype" )
colnames(sum_aov_16.ma)[4] <- "group_lettering"

p16.ma <- ggplot(sum_aov_16.ma, aes(x=reorder(genotype, -hsa.miR.16.5p), y=hsa.miR.16.5p, fill=genotype)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(hsa.miR.16.5p-sd,0), ymax=hsa.miR.16.5p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = hsa.miR.16.5p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-16-5p") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-16-5p Count")
  

p16.ma

96-5p Microarray

# Compute the analysis of variance
res.aov96.ma <- aov( hsa.miR.96.5p ~ genotype, data = newdf)
# Summary of the analysis
sum_aov_96.ma <- data_summary(newdf,varname = "hsa.miR.96.5p", groupnames = "genotype")
tukey96.ma <- TukeyHSD(res.aov96.ma)
summary(res.aov96.ma)
##             Df Sum Sq Mean Sq F value     Pr(>F)    
## genotype     4  26156    6539   28.99 0.00000438 ***
## Residuals   12   2707     226                       
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering96.ma <- multcompLetters4(res.aov96.ma,tukey96.ma) #
group_lettering96.ma
## $genotype
## CONTROL   USH1D   USH3A   USH1B   USH2A 
##     "a"     "b"     "b"     "b"     "b"
group_lettering96.ma <- data.frame(group_lettering96.ma$genotype$Letters)
group_lettering96.ma$genotype <- rownames(group_lettering96.ma)
sum_aov_96.ma <- merge(sum_aov_96.ma,group_lettering96.ma, by = "genotype" )
colnames(sum_aov_96.ma)[4] <- "group_lettering"

p96.ma <- ggplot(sum_aov_96.ma, aes(x=reorder(genotype, -hsa.miR.96.5p), y=hsa.miR.96.5p, fill=genotype)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(hsa.miR.96.5p-sd,0), ymax=hsa.miR.96.5p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = hsa.miR.96.5p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-96-5p") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-96-5p Count")
  

p96.ma

182-5p Microarray

# Compute the analysis of variance
res.aov182.ma <- aov( hsa.miR.182.5p ~ genotype, data = newdf)
# Summary of the analysis
sum_aov_182.ma <- data_summary(newdf,varname = "hsa.miR.182.5p", groupnames = "genotype")
tukey182.ma <- TukeyHSD(res.aov182.ma)
summary(res.aov182.ma)
##             Df Sum Sq Mean Sq F value   Pr(>F)    
## genotype     4   9904  2475.9   15.34 0.000115 ***
## Residuals   12   1936   161.4                     
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering182.ma <- multcompLetters4(res.aov182.ma,tukey182.ma) #
group_lettering182.ma
## $genotype
## CONTROL   USH1D   USH3A   USH1B   USH2A 
##     "a"     "b"     "b"     "b"     "b"
group_lettering182.ma <- data.frame(group_lettering182.ma$genotype$Letters)
group_lettering182.ma$genotype <- rownames(group_lettering182.ma)
sum_aov_182.ma <- merge(sum_aov_182.ma,group_lettering182.ma, by = "genotype" )
colnames(sum_aov_182.ma)[4] <- "group_lettering"

p182.ma <- ggplot(sum_aov_182.ma, aes(x=reorder(genotype, -hsa.miR.182.5p), y=hsa.miR.182.5p, fill=genotype)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(hsa.miR.182.5p-sd,0), ymax=hsa.miR.182.5p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = hsa.miR.182.5p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-182-5p") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-182-5p Count")
  

p182.ma

183-5p Microarray

# Compute the analysis of variance
res.aov183.ma <- aov( hsa.miR.183.5p ~ genotype, data = newdf)
# Summary of the analysis
sum_aov_183.ma <- data_summary(newdf,varname = "hsa.miR.183.5p", groupnames = "genotype")
tukey183.ma <- TukeyHSD(res.aov183.ma)
summary(res.aov183.ma)
##             Df Sum Sq Mean Sq F value       Pr(>F)    
## genotype     4  20327    5082   59.71 0.0000000805 ***
## Residuals   12   1021      85                         
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering183.ma <- multcompLetters4(res.aov183.ma,tukey183.ma) #
group_lettering183.ma
## $genotype
## CONTROL   USH1D   USH3A   USH2A   USH1B 
##     "a"     "b"     "b"     "b"     "b"
group_lettering183.ma <- data.frame(group_lettering183.ma$genotype$Letters)
group_lettering183.ma$genotype <- rownames(group_lettering183.ma)
sum_aov_183.ma <- merge(sum_aov_183.ma,group_lettering183.ma, by = "genotype" )
colnames(sum_aov_183.ma)[4] <- "group_lettering"

p183.ma <- ggplot(sum_aov_183.ma, aes(x=reorder(genotype,-hsa.miR.183.5p), y=hsa.miR.183.5p, fill=genotype)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(hsa.miR.183.5p-sd,0), ymax=hsa.miR.183.5p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = hsa.miR.183.5p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-183-5p") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-183-5p Count")
  

p183.ma

150-5p Microarray

# Compute the analysis of variance
res.aov150.ma <- aov( hsa.miR.150.5p ~ genotype, data = newdf)
# Summary of the analysis
sum_aov_150.ma <- data_summary(newdf,varname = "hsa.miR.150.5p", groupnames = "genotype")
tukey150.ma <- TukeyHSD(res.aov150.ma)
summary(res.aov150.ma)
##             Df  Sum Sq Mean Sq F value    Pr(>F)    
## genotype     4 5915775 1478944    19.4 0.0000358 ***
## Residuals   12  914864   76239                      
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering150.ma <- multcompLetters4(res.aov150.ma,tukey150.ma) #
group_lettering150.ma
## $genotype
##   USH2A   USH3A   USH1D   USH1B CONTROL 
##     "a"     "b"     "b"     "b"     "b"
group_lettering150.ma <- data.frame(group_lettering150.ma$genotype$Letters)
group_lettering150.ma$genotype <- rownames(group_lettering150.ma)
sum_aov_150.ma <- merge(sum_aov_150.ma,group_lettering150.ma, by = "genotype" )
colnames(sum_aov_150.ma)[4] <- "group_lettering"

p150.ma <- ggplot(sum_aov_150.ma, aes(x=reorder(genotype, +hsa.miR.150.5p), y=hsa.miR.150.5p, fill=genotype)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(hsa.miR.150.5p-sd,0), ymax=hsa.miR.150.5p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = hsa.miR.150.5p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-150-5p") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-150-5p Count")
  

p150.ma

155-5p Microarray

# Compute the analysis of variance
res.aov155.ma <- aov( hsa.miR.155.5p ~ genotype, data = newdf)
# Summary of the analysis
sum_aov_155.ma <- data_summary(newdf,varname = "hsa.miR.155.5p", groupnames = "genotype")
tukey155.ma <- TukeyHSD(res.aov155.ma)
summary(res.aov155.ma)
##             Df    Sum Sq   Mean Sq F value       Pr(>F)    
## genotype     4 633349839 158337460   70.56 0.0000000311 ***
## Residuals   12  26928266   2244022                         
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering155.ma <- multcompLetters4(res.aov155.ma,tukey155.ma) #
group_lettering155.ma
## $genotype
##   USH1B   USH3A   USH2A   USH1D CONTROL 
##     "a"     "a"     "a"     "b"     "c"
group_lettering155.ma <- data.frame(group_lettering155.ma$genotype$Letters)
group_lettering155.ma$genotype <- rownames(group_lettering155.ma)
sum_aov_155.ma <- merge(sum_aov_155.ma,group_lettering155.ma, by = "genotype" )
colnames(sum_aov_155.ma)[4] <- "group_lettering"

p155.ma <- ggplot(sum_aov_155.ma, aes(x=reorder(genotype,+hsa.miR.155.5p), y=hsa.miR.155.5p, fill=genotype)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(hsa.miR.155.5p-sd,0), ymax=hsa.miR.155.5p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = hsa.miR.155.5p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-155-5p") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-155-5p Count")
  

p155.ma

7a-5p Microarray

# Compute the analysis of variance
res.aov7a.ma <- aov( hsa.let.7a.5p ~ genotype, data = newdf)
# Summary of the analysis
sum_aov_7a.ma <- data_summary(newdf,varname = "hsa.let.7a.5p", groupnames = "genotype")
tukey7a.ma <- TukeyHSD(res.aov7a.ma)
summary(res.aov7a.ma)
##             Df   Sum Sq  Mean Sq F value       Pr(>F)    
## genotype     4 43804241 10951060   62.89 0.0000000599 ***
## Residuals   12  2089605   174134                         
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering7a.ma <- multcompLetters4(res.aov7a.ma,tukey7a.ma) #
group_lettering7a.ma
## $genotype
##   USH2A   USH3A   USH1B   USH1D CONTROL 
##     "a"     "a"     "b"     "c"     "c"
group_lettering7a.ma <- data.frame(group_lettering7a.ma$genotype$Letters)
group_lettering7a.ma$genotype <- rownames(group_lettering7a.ma)
sum_aov_7a.ma <- merge(sum_aov_7a.ma,group_lettering7a.ma, by = "genotype" )
colnames(sum_aov_7a.ma)[4] <- "group_lettering"

p7a.ma <- ggplot(sum_aov_7a.ma, aes(x=reorder(genotype, +hsa.let.7a.5p), y=hsa.let.7a.5p, fill=genotype)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(hsa.let.7a.5p-sd,0), ymax=hsa.let.7a.5p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = hsa.let.7a.5p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "let-7a-5p") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("let-7a-5p Count")
  

p7a.ma

363-3p Microarray

# Compute the analysis of variance
res.aov363.ma <- aov( hsa.miR.363.3p ~ genotype, data = newdf)
# Summary of the analysis
sum_aov_363.ma <- data_summary(newdf,varname = "hsa.miR.363.3p", groupnames = "genotype")
tukey363.ma <- TukeyHSD(res.aov363.ma)
summary(res.aov363.ma)
##             Df Sum Sq Mean Sq F value       Pr(>F)    
## genotype     4 810706  202676    66.1 0.0000000451 ***
## Residuals   12  36792    3066                         
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering363.ma <- multcompLetters4(res.aov363.ma,tukey363.ma) #
group_lettering363.ma
## $genotype
##   USH1B   USH1D   USH3A   USH2A CONTROL 
##     "a"     "b"     "c"     "c"     "d"
group_lettering363.ma <- data.frame(group_lettering363.ma$genotype$Letters)
group_lettering363.ma$genotype <- rownames(group_lettering363.ma)
sum_aov_363.ma <- merge(sum_aov_363.ma,group_lettering363.ma, by = "genotype" )
colnames(sum_aov_363.ma)[4] <- "group_lettering"

p363.ma <- ggplot(sum_aov_363.ma, aes(x=reorder(genotype,+hsa.miR.363.3p), y=hsa.miR.363.3p, fill=genotype)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(hsa.miR.363.3p-sd,0), ymax=hsa.miR.363.3p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = hsa.miR.363.3p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-363-3p") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-363-3p Count")
  

p363.ma

223-3p Microarray

# Compute the analysis of variance
res.aov223.ma <- aov( hsa.miR.223.3p ~ genotype, data = newdf)
# Summary of the analysis
sum_aov_223.ma <- data_summary(newdf,varname = "hsa.miR.223.3p", groupnames = "genotype")
tukey223.ma <- TukeyHSD(res.aov223.ma)
summary(res.aov223.ma)
##             Df Sum Sq Mean Sq F value      Pr(>F)    
## genotype     4 344295   86074    84.6 0.000000011 ***
## Residuals   12  12209    1017                        
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering223.ma <- multcompLetters4(res.aov223.ma,tukey223.ma) #
group_lettering223.ma
## $genotype
##   USH2A   USH3A   USH1D   USH1B CONTROL 
##     "a"     "b"     "c"     "c"     "c"
group_lettering223.ma <- data.frame(group_lettering223.ma$genotype$Letters)
group_lettering223.ma$genotype <- rownames(group_lettering223.ma)
sum_aov_223.ma <- merge(sum_aov_223.ma,group_lettering223.ma, by = "genotype" )
colnames(sum_aov_223.ma)[4] <- "group_lettering"

p223.ma <- ggplot(sum_aov_223.ma, aes(x=reorder(genotype,+hsa.miR.223.3p), y=hsa.miR.223.3p, fill=genotype)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(hsa.miR.223.3p-sd,0), ymax=hsa.miR.223.3p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = hsa.miR.223.3p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-223-3p") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-223-3p Count")
  

p223.ma

142-3p Microarray

# Compute the analysis of variance
res.aov142.ma <- aov( hsa.miR.142.3p ~ genotype, data = newdf)
# Summary of the analysis
sum_aov_142.ma <- data_summary(newdf,varname = "hsa.miR.142.3p", groupnames = "genotype")
tukey142.ma <- TukeyHSD(res.aov142.ma)
summary(res.aov142.ma)
##             Df     Sum Sq    Mean Sq F value      Pr(>F)    
## genotype     4 8425580545 2106395136   56.73 0.000000108 ***
## Residuals   12  445562563   37130214                        
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
group_lettering142.ma <- multcompLetters4(res.aov142.ma,tukey142.ma) #
group_lettering142.ma
## $genotype
##   USH3A   USH2A   USH1B   USH1D CONTROL 
##     "a"     "a"     "b"    "bc"     "c"
group_lettering142.ma <- data.frame(group_lettering142.ma$genotype$Letters)
group_lettering142.ma$genotype <- rownames(group_lettering142.ma)
sum_aov_142.ma <- merge(sum_aov_142.ma,group_lettering142.ma, by = "genotype" )
colnames(sum_aov_142.ma)[4] <- "group_lettering"

p142.ma <- ggplot(sum_aov_142.ma, aes(x=reorder(genotype, +hsa.miR.142.3p), y=hsa.miR.142.3p, fill=genotype)) + 
  geom_bar(stat="identity", color="black", 
           position=position_dodge(),width = 0.5) +
  geom_errorbar(aes(ymin=pmax(hsa.miR.142.3p-sd,0), ymax=hsa.miR.142.3p+sd), width=.2,
                 position=position_dodge(.9)) +
  scale_fill_manual("Genotype",values = c("CONTROL"="#374E55FF","USH1B"="#DF8F44FF","USH1D"="#00A1D5FF", "USH2A"="#B24745FF","USH3A"="#79AF97FF"))+
  geom_text(aes(label = group_lettering, y = hsa.miR.142.3p + sd), vjust=-0.4, position=position_dodge(0.9))+
  labs(title = "miRNA-142-3p") +
  theme(plot.title = element_text(hjust = 0.5),
        legend.position="none",
        panel.border = element_blank(), 
        panel.grid.major = element_blank(),
        panel.grid.minor = element_blank(), 
        axis.line = element_line(colour = "black")) +
  xlab("") +
  ylab("miRNA-142-3p Count")
  

p142.ma

microarray anova combined

down_anovas.ma <- ggarrange(p28.ma,p96.ma,p182.ma,p183.ma,p16.ma,p19b.ma,
          nrow = 2,
          ncol = 3,
          labels = "AUTO",
          legend = "none")
down_anovas.ma

#ggsave(down_anovas.ma, filename = "microarray_down.pdf", device = "pdf", width = 10, height = 10)



up_anovas.ma <- ggarrange(p363.ma,p223.ma,p150.ma,p155.ma,p7a.ma,p142.ma,
          nrow = 2,
          ncol = 3,
          labels = "AUTO",
          legend = "none")
up_anovas.ma

#ggsave(up_anovas.ma, filename = "microarray_up.pdf", device = "pdf", width = 10, height = 10)